KR20010070841A - Method for Extraction, Isolation and Identification of trans-Resveratrol and Its Derivatives with Anticarcinogenic and Antimutagenic Activity from Paeonia lactiflora Seeds - Google Patents

Method for Extraction, Isolation and Identification of trans-Resveratrol and Its Derivatives with Anticarcinogenic and Antimutagenic Activity from Paeonia lactiflora Seeds Download PDF

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KR20010070841A
KR20010070841A KR1020010033032A KR20010033032A KR20010070841A KR 20010070841 A KR20010070841 A KR 20010070841A KR 1020010033032 A KR1020010033032 A KR 1020010033032A KR 20010033032 A KR20010033032 A KR 20010033032A KR 20010070841 A KR20010070841 A KR 20010070841A
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resveratrol
viniferin
methanol
cancer
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최상원
김종성
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김효진
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Abstract

PURPOSE: A method for extracting resvedratrol and derivatives thereof having high anticancer and antimutagenic effect from a Paeoniae radix seed is provided. Whereby, the compound can be used for a medicine for prevention and treatment of cancer and functional food material. CONSTITUTION: This resvedratrol and derivatives thereof are extracted by the process consisting of: removing grease from dried Paeoniae radix seed powder by adding n-hexane; concentrating under reduced pressure at room temperature after adding methanol to the defatted Paeoniae radix seed; obtaining an extract by column chromatography; and recrystallizing after separating using a sepadex LH-20 and fraction-high speed liquid chromatography.

Description

작약씨로부터 항암 및 항돌연변이성 트란스-레즈베라트롤 및 그 유도체의 추출방법{Method for Extraction, Isolation and Identification of trans-Resveratrol and Its Derivatives with Anticarcinogenic and Antimutagenic Activity from Paeonia lactiflora Seeds}Method for Extraction, Isolation and Identification of trans-Resveratrol and Its Derivatives with Anticarcinogenic and Antimutagenic Activity from Paeonia lactiflora Seeds}

본 발명은 작약씨로부터 항암 및 항돌연변이작용이 강한 트란스-레즈베라트롤 및 그 유도체의 추출방법에 관한 것이다. 더욱 상세하게는, 작약씨로부터 암 예방 및 치료제로 유용한 트란스-레즈베라트롤(trans-resveratrol), 트란스-레즈베라트롤-4'-O-베타-D-글루코사이드(trans-resveratrol-4'-O-β-D-glucoside), 시스- & 트란스-비니페린(cis- &trans-viniferin), 스푸루티코솔(suffruticosol) A & B 성분의 추출방법에 관한 것이다.The present invention relates to a method for extracting trans-resveratrol and its derivatives, which have strong anticancer and antimutagenic activity from peony seed. More specifically, useful trans from peony seeds in cancer prevention and treatment - reds vera trawl (trans -resveratrol), trans- Reds vera trawl -4'- O-beta -D- glucoside (trans -resveratrol-4'- O -β-D-glucoside), cis-and trans relates to -viniferin), the switch extracts puru Tycho brush (suffruticosol) a & B components way - and trans- beanie Perrin (cis.

작약(芍藥, 영명: Peony, 생약명:Paeoniae radix)은 미나리아재비과 (Ranuculaceae) 작약속(Paeonia)에 속하는 약용작물로서 초본인 작약과 목본인 모란으로 구분(윤평섭, 한국원예식물도감, 1989)된다. 페오니아(Paeonia)란 이름은 그리스 신화에 나오는 페온(Paeon)신으로부터 유래되었으며, 그 기원은 중국으로 전하며, 그 외 북아프리카, 시베리아, 남중유럽 및 남아메리카에 이르기까지 전 세계적으로 32종이 널리 분포(Yamamoto,Biotechnology in Agriculture and Forestry, 4: 464-483, 1988)되어 있다.Peony (芍藥, 0 people: Peony, saengyakmyeong: Paeoniae radix) is Ranunculaceae (Ranuculaceae) peony as medicinal plants belonging to (Paeonia) separated by herbaceous of peony and neck, I Moran (yunpyeongseop, Korea horticultural plants Encyclopedia, 1989). The name Peonia originated from the Greek god, Peon, whose origin originates in China, and 32 species are distributed throughout the world, from North Africa, Siberia, South Central Europe, and South America (Yamamoto). , Biotechnology in Agriculture and Forestry , 4: 464-483, 1988).

작약은 식물 분류면에서 적작약(Paeonia lactifloraPall.), 백작약(Paeonia obovataMax.) 및 산작약(Paeonia japonicaMiyabe et Takeda)으로 구분되며, 국내에서는 적작약의 변종이 주로 재배(윤평섭, 한국원예식물도감, 1989)되고 있다.In terms of plant classification, peony is divided into Paeonia lactiflora Pall., Paeonia obovata Max. And Paeonia japonica Miyabe et Takeda. 1989).

작약은 국내에서 인삼, 당귀 다음으로 가장 많이 이용되고 있는 약용작물로서 첩약을 비롯한 쌍화탕, 사물탕, 당귀작약산, 계지가작약탕, 작약감초탕, 우황청심환, 소청룡탕 등의 생약제제의 원료로서 널리 이용되고 있다(본초학, 이상인, 1980). 작약의 국내 재배 규모는 더덕, 당귀, 도라지, 두충, 황기와 더불어 6대 주요 약용작물로서 그 생산량은 2,062 M/T (1998년 기준)으로 다른 약용작물에 비해 그 수요량이 큰 작물중의 하나이다(특용작물생산실적, 농림부통계, 1999).Peony is the most widely used medicinal crop after ginseng and tangui in Korea. (Herbology, Lee Sang-in, 1980). Peony's domestic cultivation is 6 major medicinal crops, along with Deodeok, Dangui, bellflower, tofu, and Astragalus, and its output is 2,062 M / T (as of 1998), one of the crops with higher demand than other medicinal crops. Special crop production performance, Ministry of Agriculture and Forestry, 1999).

작약뿌리는 예로부터 한방에서 진통, 진경, 진해, 부인약, 고혈압, 복통, 및 염증 치료제(Takagiet and Harada,Yakugaku Zasshi, 69: 887-892, 1969a; 69: 893-898, 1969b; Takeda et al.,J. Pharm. Pharmacol. 47: 1036-1040, 1995; Goto et al.,Planta Medica62: 436-439, 1996)로 이용되어져 왔으며, 그 주된 생리활성물질로서 페오니프로린(paeoniflorin), 옥시페오니프로린(oxypaeoniflorin), 벤조일페오니프로린(benzoylpaeoniflorin) 및 알비노프로린(albinoflorin)을 포함한 여러 가지의 모노터펜 글루코사이드(monoterpene glucosides)(Hattori et al.,Chem. Pharm. Bull. 33: 3838-3846, 1985; Kang et al.,Kor. J. Pharmacogn. 24: 247-250, 1993; Okubo et al.,Biol. Pharm. Bull. 23: 199-203, 2000)와 탄닌 및 페놀산(Sakai et al.,Eisei Kagaku35: 433-443, 1989; Goto et a., Planta Medica 62: 436-439, 1996; Satoh et al.,Biochem. Pharmacol. 53: 611-614, 1997; Tanaka et al.,Chem. Pharm. Bull. 45: 1891-1897, 1997; Tanaka et al.,Chem. Pharm. Bull. 48: 201-207, 2000)등이 알려져 있다.Peony roots are traditionally used to treat analgesic, cramping, antitussive, gynecological, high blood pressure, abdominal pain, and inflammation (Takagiet and Harada, Yakugaku Zasshi , 69: 887-892, 1969a; 69: 893-898, 1969b; Takeda et al. , J. Pharm. Pharmacol . 47: 1036-1040, 1995; Goto et al., Planta Medica 62: 436-439, 1996), and the main bioactive substances are paeoniflorin, oxy Various monoterpene glucosides including oxypaeoniflorin, benzoylpaeoniflorin and albinoflorin (Hattori et al., Chem. Pharm. Bull . 33: 3838-3846, 1985; Kang et al., Kor. J. Pharmacogn . 24: 247-250, 1993; Okubo et al., Biol. Pharm. Bull . 23: 199-203, 2000) and tannins and phenolic acids (Sakai et al. , Eisei Kagaku 35: 433-443, 1989 ; Goto et a, Planta Medica 62: 436-439, 1996; Satoh et al, Biochem Pharmacol 53:..... 611-614, 1997; Tanaka et al, Chem. Pharm.Bull. 45: 1891-1897, 199 7; Tanaka et al., Chem. Pharm. Bull . 48: 201-207, 2000).

이와같이 지금까지 작약뿌리로부터 여러 가지 생리활성물질의 검색 및 그들의 생리적·약리적 작용에 관한 많은 연구가 수행되어 온 반면, 작약씨에 관한 연구는 거의 없는 실정이다.As such, many studies have been conducted on the search for various biologically active substances and their physiological and pharmacological effects from the roots of the peony, and there are few studies on the peony seed.

레즈베라트톨(resveratrol,trans-3,4',5-trihydroxystilbene)은 나자식물 및 쌍자엽식물을 포함한 72종의 식물(31속, 12과)에 널리 분포되어 있는 피토알렉신(phytoalexin) 화합물(Langcake and Pryce,Physiol. Planta Pathol., 9: 77-86, 1976; Langcake and Pryce,Experientia33: 151-152, 1977)로써, 항암(Jang et al.,Sci., 275: 218-220, 1997; Nielsen et al.,Biochem. Biphys. Res. Commun. 275: 804-809, 2000; Schneider et al.,Cancer Lett., 158: 85-91, 2000) 뿐만 아니라 항혈전(Chung et al.,Planta Med., 58: 274-276, 1992; Frankel et al.,Lancet, 341: 1103-1104, 1993; Bertrlli et al.,Int. J. Tissue React., 17: 1-3, 1995; Pae-Asciak et al.,Clin. Chim. Acta., 235: 207-219, 1995), 항염증(Kimura et al.,Biochim. Biophys. Acta., 834: 275-278, 1985), 항고지혈증(Arichi et al,Chem. Pharm. Bull., 30: 1766-1770, 1982) 및 항산화(Burkitt and Duncan,Arch Biochem. Biophys., 381: 253-263, 2000; Tadolini et al.,Free Radic Res., 33: 105-114, 2000) 작용이 있는 생리활성성분으로써 크게 주목을 받고 있다.Resveratrol ( trans -3,4 ', 5-trihydroxystilbene) is a phytoalexin compound (Langcake) that is widely distributed in 72 plants (genus 31 and 12) including palm and dicotyledonous plants. and Pryce, Physiol.Planta Pathol ., 9: 77-86, 1976; Langcake and Pryce, Experientia 33: 151-152, 1977, anticancer (Jang et al., Sci ., 275: 218-220, 1997; Nielsen et al., Biochem.Biphys.Res.Commun. 275: 804-809, 2000; Schneider et al., Cancer Lett ., 158: 85-91, 2000) as well as antithrombotic (Chung et al., Planta Med) , 58: 274-276, 1992; Frankel et al., Lancet , 341: 1103-1104, 1993; Bertrlli et al., Int. J. Tissue React ., 17: 1-3, 1995; Pae-Asciak et al., Clin. Chim. Acta ., 235: 207-219, 1995), anti-inflammatory (Kimura et al., Biochim. Biophys. Acta ., 834: 275-278, 1985), antihyperlipidemia (Arichi et al, Chem. Pharm. Bull ., 30: 1766-1770, 1982) and antioxidants (Burkitt and Duncan, Arch Biochem. Biophys ., 381: 253-263, 2000; Tadolini et al., Free Radic Res ., 33: 10 5-114, 2000) has attracted great attention as a bioactive component with action.

피토알렉신(phytoalexin)은 식물이 어떤 환경적인 스트레스를 받거나 곰팡이와 같은 미생물이 침입할 때 자신의 몸을 보호하기 위해 생산하는 일종의 방어물질로써 포도에서 발견된 레즈베라트롤과 그 이량체(dimer)인 비니페린(viniferin)이 대표적인 화합물(Langcake and Pryce,Physiol. Plant Pathol., 9: 77-86, 1976; Langcake and Pryce,Experientia33: 151-152, 1977; Langcake and Pryce,Phytochem., 16: 1193-1196, 1977; Creasy and Coffee,J. Am. Soc. Hortic. Sci. 113: 230-234, 1988; Jeandel et al.,Am. J. Enol. Vitic. 42: 41-46, 1991)이다. 이러한 레즈베라트롤 및 그 유도체는 땅콩(Sanders et al.,J. Agric. Food Chem., 48: 1243-1246, 2000) 및 오디(Ingham,Phytochem., 15: 1791-1793, 1976) 등의 식품 뿐만 아니라 호장근(Kimura et al.,Planta Med., 49: 51-54, 1983; Shin et al.,Biochem. Biophys. Res. Commun. 243: 801-803, 1998), 상백피(Shim et al.,Biochem. Biophys. Res. Commun., 243(3): 801-803, 1998), 대황(Iida et al.,Planta Med., 61: 425-428, 1995; Shin et al.,Planta Med., 64: 283-284, 1998; Cheong et al.,Planta Med., 64: 577-578, 1998), 하수오(Martin and Roemer,Monogr. Allergy12: 145-149, 1977), 솔잎(Schoppner and Kindl,FEBS Lett. 108: 349-352, 1979) 및 그 외 식물(Prakash et al.,Phytochem. 20: 1455-1456, 1981; Kashiwada et al.,Chem. Pharm. Bull., 32: 3501-3517, 1984; Prakash et al.,Phytochem. 24: 622-624, 1985; Jayatilake et al.,J. Natural Products, 56: 1805-1810, 1993)에서도 발견되고 있다.Phytoalexin is a type of defense material produced by plants to protect their bodies in the event of any environmental stress or invasion of microorganisms, such as fungi, and resveratrol and its dimers found in grapes. Viniferin is a representative compound (Langcake and Pryce, Physiol.Plant Pathol ., 9: 77-86, 1976; Langcake and Pryce, Experientia 33: 151-152, 1977; Langcake and Pryce, Phytochem ., 16: 1193-1196, 1977; Creasy and Coffee, J. Am. Soc. Hortic. Sci . 113: 230-234 , 1988; Jeandel et al., Am. J. Enol. Vitic . 42: 41-46, 1991). . Such resveratrol and its derivatives include foods such as peanuts (Sanders et al., J. Agric. Food Chem ., 48: 1243-1246, 2000) and Audi (Ingham, Phytochem ., 15: 1791-1793, 1976). As well as Kimura et al., Planta Med ., 49: 51-54, 1983; Shin et al., Biochem. Biophys. Res. Commun . 243: 801-803, 1998), Shim et al. ....., Biochem Biophys Res Commun, 243 (3): 801-803, 1998), rhubarb (Iida et al, Planta Med, 61:... 425-428, 1995; Shin et al, Planta Med, 64: 283-284, 1998; Cheong et al., Planta Med ., 64: 577-578, 1998), Martin and Roemer, Monogr.Allergy 12: 145-149, 1977, Pine needles (Schoppner and Kindl, FEBS Lett . 108: 349-352, 1979) and other plants (Prakash et al., Phytochem . 20: 1455-1456, 1981; Kashiwada et al., Chem. Pharm. Bull ., 32: 3501-3517, 1984 Prakash et al., Phytochem . 24: 622-624, 1985; Jayatilake et al., J. Natural Products , 56: 1805-1810, 1993).

이들 식물체에서 레즈베라트롤은 유리상태로 존재하기도 하나 대부분 당류와 결합된 배당체 형태로 존재(Nonaka et al.,Chem. Pharm. Bull. 25: 2300-2305, 1977; Gorham,Prog. Phytochem., 6: 203-209, 1980)한다. 그리고 레즈베라트롤은 시스(cis)형과 트란스(trans)형이 존재하며, 자연계에서는 몇몇을 제외하고는 대부분 안정한 트란스(trans) 이성질체로 존재(Trela and Waterhouse,J. Agric. Food Chem., 44: 1253-1257, 1996)한다.In these plants, resveratrol exists in the free state, but mostly in the form of glycosides associated with sugars (Nonaka et al.,Chem. Pharm. Bull. 25: 2300-2305, 1977; Gorham,Prog. Phytochem, 6: 203-209, 1980). And resveratrol is sheathcisType and trans (trans), And in nature, except for some, most stable transtrans) Exist as isomers (Trela and Waterhouse,J. Agric. Food chem, 44: 1253-1257, 1996).

한편, 최근 레즈베라트롤은 여성호르몬 에스트로겐 유사작용이 있는 피토에스트로겐(phytoestrogen) 화합물로써(Gehm et al.,Proc. Natl. Acad. Sci. 94: 14138-14143, 1997) 새로이 주목을 받고 있다. 피토에스트로겐은 여성호르몬 에스트로겐과 유사한 분자구조를 갖고 있는 식물유래의 페놀화합물로써(Shutt and Cox,J. Endocrinol, 52: 299-310, 1972; Tang and Adams,J. Endocrinol,85: 291-297, 1980; Setchell and Adlercreutz, Academic Press Ltd, 315-345, 1988), 콩의 이소플라본(isoflavone; 제니스테인)과 아마인의 리그난(lignan; 마타이레시놀) 성분이 가장 잘 알려져 있다(Martin et al.,Endocrinology103: 1860-1867, 1978; Tang and Adams,J. Endocrinol,85: 291-297, 1980; Hutchins et al.,J. Am. DietAssoc. 95: 545-551, 1995). 이들 피토에스트로겐 화합물은 호르몬 의존성 암(유방암, 자궁암 및 전립선암)을 예방할 뿐 아니라 갱년기 여성의 에스트로겐 분비 결핍에 따른 심장병 및 골다공증의 발병을 억제하는 것으로 밝혀지고 있다(Peterson and Barnes,Biochem Biophys Res Commun. 179: 661-667, 1991; Draper et al.,J. Nutr. 127: 1795-1799, 1997; Mizutani, K. et al.,Biochem. Biophys. Res. Commun. 253: 859-863, 1998; Adlercreutz,Baillieres Clin. Endocrinol. Metab.12: 605-623, 1998; Mizutani, K. et al.,J. Nutr. Sci. Vitaminol. 46: 78-83, 2000). 이는 레즈베라트롤 및 그 유도체를 함유하고 있는 적포도주를 즐겨 마시는 사람은 심장병에 의한 사망률이 낮다는 사실(Goldberg et al.,Am. J. Enol. Vitic., 46: 159-165, 1995; Jeandet et al.,J. Phytopathology143: 135-139, 1995)을 뒷받침해 주고 있다. 또한, 레즈베라트롤 유도체는 피부 멜라닌(melanin) 색소의 합성을 촉진하는 티로시나제(tyrosinase) 효소의 강력한 저해제로써 피부 미백효과가 우수한 화장품의 신소재(Iida et al.,Planta Med., 61: 425-428, 1995; Shimizu et al.,Planta Med., 64: 408-412, 1998; Shin et al.,Biochem. Biophys. Res. Commun. 243: 801-803, 1998)로써 크게 각광받고 있으며, 아울러 항알레르기 작용(Cheong et al.,Planta Med., 65: 266-268, 1999)이 있는 것으로 밝혀지고 있다.Recently, resveratrol is attracting new attention as a phytoestrogen compound having a female hormone estrogen-like action (Gehm et al., Proc. Natl. Acad. Sci . 94: 14138-14143, 1997). Phytoestrogens are plant-derived phenolic compounds with molecular structures similar to female hormone estrogens (Shutt and Cox, J. Endocrinol , 52: 299-310, 1972; Tang and Adams, J. Endocrinol, 85: 291-297, 1980; Setchell and Adlercreutz, Academic Press Ltd, 315-345, 1988), isoflavones (genistein) and lignans (mattayresinol) of flax seed are best known (Martin et al., Endocrinology) 103: 1860-1867, 1978; Tang and Adams, J. Endocrinol, 85: 291-297, 1980; Hutchins et al., J. Am. DietAssoc . 95: 545-551, 1995). These phytoestrogens compounds have been shown to prevent hormone-dependent cancers (breast cancer, uterine cancer and prostate cancer) as well as to suppress the development of heart disease and osteoporosis due to estrogen secretion deficiency in menopausal women (Peterson and Barnes, Biochem Biophys Res Commun . 179: 661-667, 1991; Draper et al., J. Nutr . 127: 1795-1799, 1997; Mizutani, K. et al., Biochem. Biophys.Res.Commun. 253: 859-863, 1998; Adlercreutz , Baillieres Clin.Endocrinol . Metab. 12: 605-623, 1998; Mizutani, K. et al., J. Nutr. Sci. Vitaminol . 46: 78-83, 2000). This indicates that people who drink red wine containing resveratrol and its derivatives have a low mortality rate from heart disease (Goldberg et al., Am. J. Enol. Vitic ., 46: 159-165, 1995; Jeandet et. al., J. Phytopathology 143: 135-139, 1995). In addition, resveratrol derivatives are potent inhibitors of tyrosinase enzymes that promote the synthesis of skin melanin pigments (Iida et al., Planta Med ., 61: 425-428). , 1995; Shimizu et al., Planta Med ., 64: 408-412, 1998; Shin et al., Biochem. Biophys.Res.Commun. 243: 801-803, 1998). Action (Cheong et al., Planta Med ., 65: 266-268, 1999).

한편, 일부의 식물에서는 레즈베라트롤 이외에 다수의 레즈베라트롤 소당체(oligostilbenes) 성분(Pryce and Langcake,Phytochem., 16: 1452-1454, 1977; Kawabata et al.,Tetrahedron Letters30: 3785-3788, 1989; Kurihara etal.,Phytochem., 30: 649-653, 1991; Sarker et al.,Tetrahedron55: 513-524, 1999)이 발견되었으며, 이들은 항균(Sotheeswaran et al.,J. Chem. Soc. Perkin Trans. 1: 159-162, 1985; Geewananda et al.,Phytochem., 25: 1498-1500, 1986; Suzuki et al.,Agric. Biol. Chem., 51: 1003-1008, 1987; Uvais et al.,Phytochem., 26: 799-801, 1987; Bokel et al.,Phytochem., 27: 377-380, 1988; Kawabata et al.,Tetrahedron Letters30: 3785-3788, 1989; Kitanaka et al.,Chem. Pharm. Bull. 38: 432-435, 1990; Kulanthaivel et al.,Planta Med., 61: 41-44, 1995) 뿐만 아니라 항염증(Lee et al.,Planta Med., 64: 204-207, 1998), 간보호(Oshima et al.,Experientia51: 63-66, 1995) 및 항호르몬(Sarker et al.,Tetrahedron55: 513-524, 1999) 작용 등이 있는 것으로 보고되고 있다. 지금까지 밝혀진 대표적인 레즈베라트롤 소당체 성분에는 이량체(dimer)인, 비니페린(viniferin)(Langcake and Pryce,Physiol. Plant Pathol., 9: 77-86, 1976; Langcake and Pryce,Experientia33: 151-152, 1977; Langcake and Pryce,Phytochem., 16: 1193-1196, 1977; Sotheeswaran et al.,J. Chem. Soc. Perkin Trans., 1: 159-162, 1985; Kurihara et al.,Agric. Biol. Chem., 54: 1097-1099, 1990), 스커푸신(scirpusin)(Nakajima et. al.,Chem. Pharm. Bull., 26: 3050-3057, 1978), 발라노카폴(balanocarpol)(Champika Diyasena et al.,J. Chem. Soc. Perkin Trans., 1: 1807-1809, 1985), 팔리돌(pallidol)(Khan et al.,Phytochem., 25: 1945-1948, 1986), 암페로프신(ampelopsin)(Oshima et al.,Tetrahedron46: 5121-5126, 1990; Oshima et al.,Tetrahedron49: 5801-5804, 1993; Oshima etal.,Experientia51: 63-66, 1995)과, 삼량체(trimer)인 알파-비니페린(α-viniferin)(Pryce and Langcake,Phytochem., 16: 1452-1454, 1977; Kitanaka et al.,Chem. Pharm. Bull., 38: 432-435, 1990; Kulanthaivel et al.,Planta Med., 61: 41-44, 1995; Lee et al.,Planta Med., 64: 204-207, 1998) 디스티콜(distichol)(Uvais et al.,Phytochem., 26: 799-801, 1987), 코팔리페롤(copalliferol)(Geewananda et al.,Phytochem., 25: 1498-1500, 1986), 스테모노포롤(stemonoporol) (Samaraweera et al.,Phytochem., 24: 2585-2587, 1982), 미야베놀(miyabenol)(Suzuki et al.,Agric. Biol. Chem., 51: 1003-1008, 1987; Kurihara et al.,Agric. Biol. Chem., 54: 1097-1099, 1990), 카나리쿨라톨(canaliculatol)(Bokel et al.,Phytochem., 27: 377-380, 1988), 다비디올(davidiol) A-C(Tanaka et al.,Phytochem., 53: 1009-1014, 2000), 바티카놀(vaticanol) A(Tanaka et al.,Phytochem., 54: 63-69, 2000)과, 사량체(tetramer)인 바티카피놀(vaticaffinol)(Sotheeswaran et al.,J. Chem. Soc. Perkin Trans., 1: 159-162, 1985), 바티카놀(vaticanol) B & C(Tanaka et al.,Phytochem., 54: 63-69, 2000), 코보페놀(kobophenol) (Kawabata et al.,Tetrahedron Letters30: 3785-3788, 1989; Kawabata et al.,Phytochem., 30: 645-647, 1991; Kurihara et al.,Phytochem., 30: 649-653, 1991), 홉페아페놀(hopeaphenol)(Champika Diyasena et al.,J. Chem. Soc. Perkin Trans., 1: 1807-1809, 1985) 및 오량체(pentamer)인 다비디올(davidiol) D (Ohyama et al.,Tetrahedron Letters37: 5155-5159, 1996) 등이 알려져 있다.On the other hand, in some plants, in addition to resveratrol, a number of components of resveratrol oligostilbenes (Pryce and Langcake, Phytochem ., 16: 1452-1454, 1977; Kawabata et al., Tetrahedron Letters 30: 3785-3788, 1989; Kurihara et al., Phytochem ., 30: 649-653, 1991; Sarker et al., Tetrahedron 55: 513-524, 1999) and these were antibacterial (Sotheeswaran et al., J. Chem. Soc. Perkin). Trans 1:. 159-162, 1985; Geewananda et al, Phytochem, 25: 1498-1500, 1986; Suzuki et al, Agric Biol Chem, 51:...... 1003-1008, 1987; Uvais et al. , Phytochem ., 26: 799-801, 1987; Bokel et al., Phytochem ., 27: 377-380, 1988; Kawabata et al., Tetrahedron Letters 30: 3785-3788, 1989; Kitanaka et al., Chem. Pharm. Bull . 38: 432-435, 1990; Kulanthaivel et al., Planta Med ., 61: 41-44, 1995) as well as anti-inflammatory (Lee et al., Planta Med ., 64: 204-207, 1998). ), Liver protection (Oshima et al., Experientia 51: 63-66, 1995) and anti-hormones (Sarker et al., Tetrahedron 55: 513-524, 1999) It is reported that there is a dragon. Representative resveratrol small-saccharide components so far identified include dimer, viniferin (Langcake and Pryce, Physiol.Plant Pathol ., 9: 77-86, 1976; Langcake and Pryce, Experientia 33: 151 -152, 1977; Langcake and Pryce, Phytochem ., 16: 1193-1196, 1977; Sotheeswaran et al., J. Chem. Soc.Perkin Trans ., 1: 159-162, 1985; Kurihara et al., Agric. Biol. Chem ., 54: 1097-1099, 1990), scirpusin (Nakajima et. Al., Chem. Pharm. Bull ., 26: 3050-3057, 1978), balanocarpol (Champika) Diyasena et al., J. Chem. Soc.Perkin Trans ., 1: 1807-1809, 1985), palidol (Khan et al., Phytochem ., 25: 1945-1948, 1986), amperofcin (ampelopsin) (Oshima et al., Tetrahedron 46: 5121-5126, 1990; Oshima et al., Tetrahedron 49: 5801-5804, 1993; Oshima etal., Experientia 51: 63-66, 1995) and trimers ( trimer) alpha-viniferin (Pryce and Langcake, Phytochem ., 16: 1452-1454, 1977; Kitanaka et al., Chem. Pharm. Bull ., 38: 432-435, 1990; Kulanthaivel et al., Planta Med ., 61: 41-44, 1995; Lee et al., Planta Med ., 64: 204-207, 1998) distichol (Uvais et al. , Phytochem ., 26: 799-801, 1987), copalliferol (Geewananda et al., Phytochem ., 25: 1498-1500, 1986), stemonoporol (Samaraweera et al., Phytochem , 24: 2585-2587, 1982), miyabenol (Suzuki et al., Agric. Biol. Chem ., 51: 1003-1008, 1987; Kurihara et al., Agric. Biol. Chem ., 54: 1097-1099, 1990), canaliculatol (Bokel et al., Phytochem ., 27: 377-380, 1988), davidiol AC (Tanaka et al., Phytochem . 53: 1009-1014, 2000), vaticanol A (Tanaka et al., Phytochem ., 54: 63-69, 2000), and the tetramer, vaticaffinol (Sotheeswaran et. al., J. Chem. Soc.Perkin Trans ., 1: 159-162, 1985), vaticanol B & C (Tanaka et al., Phytochem ., 54: 63-69, 2000), cobophenols (kobophenol) (Kawabata et al., Tetrahedron Letters 30: 3785-3788, 1989; Kawabata et al., Phytochem ., 30: 645-647, 1991; Kurihara et al., Phytochem ., 30: 649-653, 1991 ), Hopaphenol (Champika Diyasena et al., J. Chem. Soc. Perkin Trans ., 1: 1807-1809, 1985) and the pentamer davidiol D (Ohyama et al. , Tetrahedron Letters 37: 5155-5159, 1996).

이들 레즈베라트롤 소량체는 지금까지 단지 5과(科)의 식물(Dipterocarpaceae,Vitaceae,Cyperaceae,Gnetaceae및Leguminosae)에서만 발견(Kawabata et al.,Tetrahedron Letters30: 3785-3788, 1989; Kurihara et al.,Phytochem., 30: 649-653, 1991)되었으나, 최근 작약속에 속하는 모란(Paeonia suffruticosa)씨에서도 처음으로 시스-레즈베라트롤(cis- resveratrol, 미량의trans-resveratrol 함유) 및 그것의 삼량체(trimer) 성분인 서프루티코솔(suffruticosol) A, B 및 C 등이 분리(Sarker et al.,Tetrahedron55: 513-524, 1999)된 바 있다. 이와같이 레즈베라트롤 및 그 유도체는 암, 고혈압, 심장병 및 골다공증 등의 여러 가지 만성질환을 예방 및 치료할 수 있는 생리활성물질로써 각광을 받고 있으며, 아울러 그들 성분을 함유한 식물은 중요한 의약자원으로서 개발할 가치가 충분하다.These resveratrol dimers have only been found so far in only five plants ( Dipterocarpaceae , Vitaceae , Cyperaceae , Gnetaceae and Leguminosae ) (Kawabata et al., Tetrahedron Letters 30: 3785-3788, 1989; Kurihara et al. , Phytochem ., 30: 649-653, 1991), but also the first peony ( Paeonia suffruticosa ), cis -resveratrol (containing traces of trans -resveratrol) and its trimers (for the first time). Supruticosol A, B and C, which are trimer components, have been isolated (Sarker et al., Tetrahedron 55: 513-524, 1999). As such, resveratrol and its derivatives are in the spotlight as bioactive substances that can prevent and treat various chronic diseases such as cancer, hypertension, heart disease and osteoporosis, and plants containing these components are worth developing as important medicinal resources. Is enough.

본 발명은 작약씨로부터 60% 및 100% 수용성 메탄올 용액으로 추출한 후, 상기 60% 및 100% 메탄올 추출물을 농축한 후 다시 다이아이온 HP-20, 실리카겔 및 세파덱스 LH-20 칼럼 및 분취-고속액체크로마토그래피를 실시하여 레즈베라트롤 및 그 유도체의 제공과 레즈베라트롤 및 그 유도체의 추출방법 제공을 목적으로 한다.The present invention extracts 60% and 100% aqueous methanol solution from Peony seed, and then concentrates the 60% and 100% methanol extract, and then diion HP-20, silica gel and Sephadex LH-20 column and preparative-high-speed liquid Chromatography is carried out to provide resveratrol and its derivatives and to provide a method for extracting resveratrol and its derivatives.

도 1은 작약씨로부터 분리된 6가지 스틸벤화합물의 고속액체크로마토그람이다.1 is a high-performance liquid chromatogram of six stilbene compounds isolated from peony seeds.

본 발명의 작약씨로부터 트란스-레즈베라트롤 및 그 유도체의 추출방법은 건조된 작약씨를 미세하게 분쇄하여 노르말 핵산으로 탈지하여 지방을 제거하는 단계와, 지방을 제거한 작약씨 잔사에 메탄올을 가하여 상온에서 추출한 후 감압·농축하는 단계와, 전기의 농축물을 다이아이온 HP-20로 칼럼크로마토그래피를 실시하여 추출물을 얻는 단계와, 전기의 추출물을 세파덱스 LH-20 칼럼 및 분취-고속액체크로마토그래피를 이용하여 분리한 후 재결정하는 단계를 포함한다.The method of extracting trans-resveratrol and its derivatives from the peony seed of the present invention comprises the steps of finely pulverizing the dried peony seed, degreasing it with normal nucleic acids to remove fat, and adding methanol to the peony seed residue from which the fat was removed at room temperature. Extracting and depressurizing and concentrating, and performing the previous step by column chromatography on diion HP-20 to obtain an extract, and extracting the former by Sephadex LH-20 column and preparative high-speed liquid chromatography. And then recrystallization using the separation.

한편 본 발명의 또다른 작약씨로부터 트란스-레즈베라트롤 및 그 유도체의 추출방법은 건조된 작약씨를 미세하게 분쇄한 후 메탄올로 추출한 후 감압·농축하는 단계와, 메탄올추출물을 다시 메탄올로 가용화시킨 후 노르말 핵산으로 탈지하여 지방을 제거하는 단계와, 전기의 메탄올추출물에 에테르 및 에틸아세테이트를 순차적으로 넣고 분획한 후 얻어지는 에테르 및 에틸아세테이트층을 감압·농축하는 단계와, 전기의 에테르 및 에틸아세테이트층을 순차적으로 실리카겔, 세파덱스 엘에이치-20 칼럼 및 분취용 고속액체크로마토그래피를 이용하여 분리한 후 재결정하는 단계를 포함한다.Meanwhile, another method of extracting trans-resveratrol and its derivatives from another peony seed of the present invention comprises finely pulverizing the dried peony seed, extracting with methanol, depressurizing and concentrating the methanol extract, and then solubilizing the methanol extract again with methanol. Degreasing with normal nucleic acid to remove fat, depressurizing and concentrating the ether and ethyl acetate layers obtained by sequentially adding and fractionating ether and ethyl acetate to the methanol extract of the former, and ether and ethyl acetate layers Sequentially separating and recrystallizing using silica gel, Sephadex L-20 column and preparative high performance liquid chromatography.

본 발명에서 사용한 메탄올의 바람직한 농도는 40% 내지 100% 수용액이며 가장 바람직하기는 80% 내지 100% 수용액이다. 이 때 추출용매를 메탄올 대신 에탄올을 사용할 수 있다. 본 발명에서 추출하는 단계에서 바람직한 온도는 20℃ 내지 60℃이며 가장 바람직하기는 50℃이다. 아울러 추출하는 모든 단계에서는 햇빛을 차단하여 실행하는 것이 좋다.Preferred concentrations of methanol used in the present invention are 40% to 100% aqueous solution, most preferably 80% to 100% aqueous solution. In this case, ethanol may be used instead of methanol as the extraction solvent. Preferred temperature in the extraction step in the present invention is 20 ℃ to 60 ℃ and most preferably 50 ℃. In addition, it is advisable to block the sunlight at all stages of extraction.

상기의 추출방법에 의해서 작약씨로부터 추출한 트란스-베즈베라트롤 및 그 유도체는 하기 구조식 Ⅰ의 트란스-베즈베라트롤(trans-Resveratrol), 구조식 Ⅱ의 트란스-레즈베라트롤-4'-글루코사이드(Resveratrol-4'-O-β-D-glucopyranoside), 구조식 Ⅲ의 트란스-비니페린(trans-ε-Viniferin), 구조식 Ⅳ의 시스-비니페린(cis-ε-Viniferin), 구조식 Ⅴ의 서프루티코솔 A(Suffruticosol A) 및 구조식 Ⅵ의 서프루티코솔 B(Suffruticosol B)이다.Trans-Besveratrol and its derivatives extracted from Peony seeds by the above extraction method are trans- Resveratrol of the following structural formula I, Trans-Resveratrol-4'-glucoside of the structural formula II (Resveratrol- 4'- O -β-D-glucopyranoside) , of formula ⅲ trans- beanie Perrin (trans--Viniferin ε), ⅳ structural formula of the cis-beanie Perrin (cis--Viniferin ε), fruity kosol standing ⅴ of formula a ( Suffruticosol A) and Suffruticosol B of formula VI.

(구조식 I) (구조식 II)(Formula I) (Formula II)

(구조식 III) (구조식 Ⅳ)(Structure III) (Structure IV)

(구조식 Ⅴ) (구조식 Ⅵ)(Structure Formula Ⅴ) (Structure Formula Ⅵ)

한편 상기의 추출방법에 의해 작약씨로부터 추출한 트란스-레즈베라트롤, 트란스-레즈베라트롤-4'-글루코사이드, 트란스-비니페린, 시스-비니페린, 서프루티코솔 A 또는 서프루티코솔 B 중에서 어느 하나의 단일 화합물을 유효성분으로 함유하는 신경교종암(C6) 치료제 조성물로 사용할 수 있으며, 또한 트란스-비니페린 또는 시스-비니페린 성분 중 어느 하나의 단일 화합물을 유효성분으로 함유하는 간암, 유방암, 대장암 또는 자궁암 치료제 조성물로 사용할 수 있다.On the other hand, any one of trans-resveratrol, trans-resveratrol-4'-glucoside, trans-viniferin, cis-viniferin, serruticosol A, or serruticosol B extracted from peony seed by the above extraction method Liver cancer, breast cancer, colorectal cancer which can be used as a therapeutic composition for glioma cancer (C6) containing a single compound as an active ingredient, and also containing a single compound of any one of trans-viniferin or cis-viniferin as an active ingredient Or as uterine cancer therapeutic composition.

본 발명에 의해 작약씨로부터 추출한 트란스-레즈베라트롤, 트란스-레즈베라트롤-4'-글루코사이드, 트란스-비니페린, 시스-비니페린, 서프루티코솔 A 또는 서프루티코솔 B를 유효성분으로 함유하는 항돌연변이제로 사용할 수 있으며, 암 예방제 또는 치료제에 있어서, 상기 추출물들을 유효성분으로 함유하는 수액제, 환제,정제 또는 캡슐제로 사용할 수 있다.Trans-Resveratrol, Trans-Resveratrol-4'-glucoside, Trans-Viniferin, Cis-Viniferin, Serfruticosol A or Serfruticosol B extracted from Peony seed according to the present invention as an active ingredient It can be used as an anti-mutant, and in cancer prevention or treatment, it can be used as an aqueous solution, a pill, a tablet or a capsule containing the extract as an active ingredient.

한편 암 예방용 식품에 있어서, 본 발명에 의해 작약씨로부터 추출한 트란스-레즈베라트롤, 트란스-레즈베라트롤-4'-글루코사이드, 트란스-비니페린, 시스-비니페린, 서프루티코솔 A 또는 서프루티코솔 B를 유효성분으로 함유한 분말차, 과립차, 음료, 정제 또는 캡슐제 형태의 건강보조식품에 이용할 수 있다.Meanwhile, in the food for cancer prevention, trans-resveratrol, trans-resveratrol-4'-glucoside, trans-viniferin, cis-viniferin, serproticosol A or surfuti extracted from peony seed according to the present invention Powdered tea, granulated tea, beverage, tablets or capsules containing Kosol B as an active ingredient can be used in the dietary supplement.

이하 본 발명의 구체적인 방법을 실시예를 들어 상세히 설명하고자 하지만 본 발명의 권리범위는 이들 실시예에만 한정되는 것은 아니다.Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

<실시예 1><Example 1>

건조 작약씨 300g을 고르게 분쇄한 후 노르말-핵산을 가하여 탈지한 다음 얻어진 탈지박에 100% 메탄올 가하여 일주일간 상온에서 3회 반복 추출한 후 여과·농축하여 메탄올추출물(25.5g)을 얻었다.The dried peony seed was crushed evenly and then degreased by adding normal-nucleic acid, 100% methanol was added to the obtained skim foil, and extracted three times at room temperature for one week, followed by filtration and concentration to obtain methanol extract (25.5g).

이 메탄올추출물에 10% 수용성 메탄올용액을 가하여 가용화한 후 미리 10% 수용성 메탄올용액으로 평형화시켜 놓은 다이아이온 HP-20 칼럼(직경 6㎝ × 길이 60㎝)에 충진하여 흡착시킨 후 20% 수용성 메탄올(5ℓ), 40% 수용성 메탄올(10ℓ) 및 80% 수용성 메탄올(10ℓ) 용액을 차례로 용리하였다.The methanol extract was solubilized by adding 10% aqueous methanol solution, and then packed into a DIION HP-20 column (6 cm in diameter x 60 cm in length), which had been equilibrated with 10% aqueous methanol solution, and adsorbed. 5 L), 40% aqueous methanol (10 L) and 80% aqueous methanol (10 L) solutions were eluted sequentially.

여기서 얻어진 40% 수용성 메탄올추출액을 감압·농축시켜 얻은 건조물질(4.82g)를 메탄올(40mL)로 녹인 후 일부(5ml)를 반복하여 미리 메탄올로평형화시켜 놓은 세파덱스 LH-20(파아마시아, 스웨덴) 칼럼(직경 2.5cm × 길이 1m)에 충진 후 같은 용매 1ℓ로 튜브당 3ml씩 200개 분획으로 나누어 용출하였다.Sepadex LH-20 (Pamacia, which was previously equilibrated with methanol by dissolving the dried material (4.82g) obtained by depressurizing and concentrating the 40% aqueous methanol extract obtained in this place with methanol (40mL) and repeating a portion (5ml). Sweden) After filling into a column (diameter 2.5 cm x length 1 m), the mixture was eluted by dividing it into 200 fractions of 3 ml per tube with 1 liter of the same solvent.

이때 자외선 검출기 필터(280㎚)를 통하여 순차적으로 분리되어 나오는 2가지 자외선 흡수분획(I, & II)을 모아 감압·농축하고 이 중 분획 I(분획 20-35)로부터 백색의 비결정 트란스-레즈베라트롤-4'-O-베타-D-글루코사이드(trans- resveratrol-4'-O-β-D-glucoside, 분자량 390) 0.25g을 얻었으며,At this time, two ultraviolet absorption fractions (I, & II), which are separated sequentially through the ultraviolet detector filter (280 nm), are collected and decompressed and concentrated, and a white amorphous trans-resvera is extracted from fraction I (fraction 20-35). 0.25 g of troll-4'- 0 -beta-D-glucoside ( trans -resveratrol-4'- 0- β-D-glucoside, molecular weight 390) was obtained.

다음 분획 II(분획 45-57)는 메탄올로 다시 녹인 후 분취-액체크로마토그래피를 반복 실시하여 연한 갈색의 무결정 서프루티코솔(suffruticosol) A(분자량 680, 분리시간 16.8분) 2.27g과 서프루티코솔 B(분자량 680, 분리시간 23.5분) 1.32g을 각각 분리하였다.Fraction II (fraction 45-57) was dissolved in methanol again and preparative-liquid chromatography was repeated. 2.27 g of light brown amorphous suffruticosol A (molecular weight 680, separation time 16.8 minutes) and sulphur 1.32 g of Corsol B (molecular weight 680, separation time 23.5 minutes) were separated, respectively.

한편 상기 분취용 액체크로마토그래피의 조건은 다음과 같다.Meanwhile, the conditions of the preparative liquid chromatography are as follows.

워터스 델타프렙-4000 고속액체크로마토그래피;Waters Delta Prep-4000 Fast Liquid Chromatography;

칼럼, RCM Novapak C18(2.5cm × 10cm);Column, RCM Novapak C 18 (2.5 cm × 10 cm);

자외선 검출기(UV280nm);Ultraviolet detector (UV 280 nm );

유속, 5.0ml/분;Flow rate, 5.0 ml / min;

용매, 0.1% 트리플로로초산 함유 20% 수용성 아세트니트릴 용액20% aqueous acetonitrile solution with solvent, 0.1% trifluoroacetic acid

한편, 위에서 얻은 80% 수용성 메탄올추출물(5.15g)을 위와 동일한 방법으로세파덱스 LH-20 칼럼크로마토그래피 및 분취-액체크로마토그래피(용매, 0.1% 트리플로로초산 함유 25% 아세트니트릴 용액)를 반복 실시하여 연한 갈색의 비결정 트란스-레즈베라트롤(trans-resveratrol, 분자량 228, 분리시간 17.6분) 0.18g, 트란스-비니페린(trans-viniferin, 분자량 484, 분리시간 23.1분) 2.48g 및 시스-비니페린(cis- viniferin, 분자량 484, 분리시간 28.7분) 1.35g을 각각 얻었다.Meanwhile, the 80% water-soluble methanol extract (5.15 g) obtained above was repeated with Sephadex LH-20 column chromatography and preparative-liquid chromatography (solvent, 25% acetonitrile solution containing 0.1% trifluoroacetic acid) in the same manner as above. Light brown amorphous trans-resveratrol ( trans -resveratrol, molecular weight 228, separation time 17.6 minutes) 0.18g, trans-viniferin ( trans -viniferin, molecular weight 484, separation time 23.1 minutes) 2.48g and cis-beanie 1.35 g of perrin ( cis -viniferin, molecular weight 484, separation time 28.7 minutes) were obtained, respectively.

<실시예 2><Example 2>

건조 작약씨 300g을 분쇄한 후 여기에 메탄올 2ℓ를 가하여 상온에서 24시간 추출·여과한다. 위의 추출조작을 3회 반복 실시하여 얻은 메탄올 혼합 추출액을 감압·농축하여 메탄올추출물(37.5g)을 얻었다.After grinding 300 g of dry peony seeds, 2 L of methanol was added thereto, followed by extraction and filtration at room temperature for 24 hours. Methanol extract (37.5 g) was obtained by depressurizing and concentrating the methanol mixture extract obtained by repeating the above extraction operation three times.

다음, 메탄올추출물에 80% 수용성 메탄올용액 500ml를 가하여 다시 녹인 후 여기에 노르말-핵산 500ml를 가하여 2회 반복 탈지한 후 얻어진 하층을 200ml까지 감압·농축한 다음 여기에 에테르 및 에틸아세테이트 1리터를 각각 넣어 순차적으로 2회 반복 분획하여 에테르추출물(7.88그람)과 에틸아세테이트 추출물(0.54g)을 각각 얻었다.Next, 500 ml of 80% water-soluble methanol solution was added to the methanol extract to dissolve again, and 500 ml of normal-nucleic acid was added thereto, followed by repeated degreasing twice. The resulting lower layer was depressurized and concentrated to 200 ml, and then 1 liter of ether and ethyl acetate were added thereto. The mixture was repeated two times in succession to obtain an ether extract (7.88 grams) and an ethyl acetate extract (0.54 g), respectively.

에테르추출물 전량을 클로로포름/메탄올(3:1, v/v) 혼액 30ml에 현탁시킨 후 일정량을 미리 같은 용매로 평형화시켜 놓은 실리카겔 60(70-130메쉬, 머어크, 독일) 700g으로 충진시킨 직경 5센티미터의 칼럼으로 옮기고 클로로포름/메탄올(3:1, v/v) 혼액 2리터로 튜브당 5ml씩 200개 분획으로 나누어 반복 용출한다.The whole ether extract was suspended in 30 ml of a chloroform / methanol (3: 1, v / v) mixture, and then filled with 700 g of silica gel 60 (70-130 mesh, Merck, Germany), which was previously equilibrated with the same solvent. Transfer to a centimeter column and repeat elution with 200 liters of 5 ml per tube with 2 liters of chloroform / methanol (3: 1, v / v) mixture.

이들 분획을 6개의 분획(분획 1, 5-15번, 0.1g; 분획 2, 16-45번, 0.59g; 분획 3, 46-78, 0.81g; 분획 4, 79-91번, 0.14g; 분획 5, 92-123, 0.62g; 분획 6, 124-167번, 3.2g)으로 나눈 후 그 중 분획 3을 모아 감압·농축한 후 다시 메탄올로 재결정하여 연한 갈색의 트란스-레즈베라트롤(trans-resveratrol, 분자량 228) 0.16g을 얻었으며, 그리고 분획 6으로부터 같은 방법으로 연한 갈색의 서프루티코솔 A(suffruticosol A, 분자량 680) 2.12g과 서프루티코솔 B(suffruticosol B, 분자량 680) 1.20g을 각각 얻었다. 다음, 분획 4와 5을 모아 감압·농축시킨 후 다시 메탄올로 녹인 다음 세파덱스 LH-20(파아마시아, 스웨덴) 100g으로 충진시킨 직경 2센티미터의 칼럼으로 옮기고 메탄올 1ℓ로 튜브당 3ml씩 200개 분획으로 나누어 용출한다. 이때 자외선 검출기 필터(280 나노미터)를 통하여 순차적으로 분리되어 나오는 비니페린 (시스- 및 트란스-비니페린) 혼합물 3.14g을 얻었다. 다음, 이 혼합물을 80% 수용성 메탄올용액으로 녹인 후 분취-액체크로마토그래피를 반복 실시하여 연한 갈색의 무결정의 트란스-비니페린(trans-viniferin, 분자량 484, 분리시간, 17.8분) 1.79g과 시스-비니페린(cis-viniferin, 분자량 454, 분리시간 22.4분) 1.07g을 각각 얻었다.These fractions were divided into six fractions (fraction 1, 5-15, 0.1 g; fraction 2, 16-45, 0.59 g; fraction 3, 46-78, 0.81 g; fraction 4, 79-91, 0.14 g; Fraction 5, 92-123, 0.62 g; Fraction 6, No. 124-167, 3.2 g), collected fraction 3, reduced pressure and concentration, and recrystallized with methanol again to give a light brown trans-resveratrol ( trans 0.16 g of -resveratrol, molecular weight 228), and 2.12 g of light brown suffruticosol A (molecular weight 680) and 1.20 g of suffruticosol B (molecular weight 680) were obtained from fraction 6 in the same manner. Respectively obtained. Then, fractions 4 and 5 were collected, decompressed and concentrated, and then dissolved in methanol. Then, the resultant was transferred to a column of 2 centimeters in diameter filled with 100 g of Sephadex LH-20 (Paamacia, Sweden) and 200 ml of 3 ml per tube with 1 liter of methanol. Dilute into fractions and elute. At this time, 3.14 g of a mixture of viiniferin (cis- and trans-viniperin), which were sequentially separated through an ultraviolet detector filter (280 nanometers), was obtained. Then, the mixture was dissolved in 80% aqueous methanol solution and preparative-liquid chromatography was repeated to yield 1.79 g of light brown amorphous trans- viniferin (molecular weight 484, separation time, 17.8 minutes) and cis-. 1.07 g of viniferin (cis-viniferin, molecular weight 454, separation time 22.4 minutes) was obtained, respectively.

상기와 동일한 방법으로 에틸아세테이트 추출물 전량을 100% 메탄올에 녹인 후 세파덱스 LH-20 칼럼크로마토그래피하여 6개분획으로 나누어 용출한 후 이 중 분획 3번을 농축하여 백색 분말의 트란스-레즈베라트롤-4'-O-베타-D-글루코사이드(trans-resveratrol-4'-O-β-D-glucoside, 분자량 390) 0.22g을 얻었다. 이때, 사용된 분취용 액체크로마토그래피(워터스 델타프렙-4000 고속액체크로마토그래피; 칼럼, RCMNovapak C18(2.5센티미터 × 10센티미터); 자외선 검출기(UV280 & 308nm); 유속, 5.0ml/분; 용매, 0.1% 트리플로로초산 함유 30% 아세트니트림 용액)로 분리하였다.In the same manner as above, the total amount of ethyl acetate extract was dissolved in 100% methanol, and then eluted by dividing into 6 fractions by Sepadex LH-20 column chromatography. The fraction 3 was concentrated to concentrate the trans-resveratrol- 4'- O - beta -D- glucoside (trans -resveratrol-4'- O -β- D-glucoside, molecular weight 390) was obtained 0.22g. At this time, the preparative liquid chromatography used (Waters Delta Prep-4000 high-performance liquid chromatography; column, RCMNovapak C 18 (2.5 centimeters × 10 centimeters); UV detector (UV 280 & 308 nm); flow rate, 5.0 ml / min; solvent , 30% acetnitrim solution containing 0.1% trifluoroacetic acid).

<실시예 3><Example 3>

건조 작약씨 5g을 분쇄한 후 여기에 메탄올 300ml를 가하여 50℃로 조정된 자석교반기에서 24시간 동안 2회 반복 추출한 후 여과 및 감압·농축하였다.5 g of dry peony seeds were pulverized, and 300 ml of methanol was added thereto, followed by repeated extraction for 24 hours in a magnetic stirrer adjusted to 50 ° C., followed by filtration, reduced pressure, and concentration.

그 후, 메탄올추출물(0.83g)을 80% 수용성메탄올 100ml로 녹인 후 여기에 노르말-핵산 300ml 가하여 2회 반복 탈지한 다음 하층을 감압·농축하였다.Thereafter, methanol extract (0.83 g) was dissolved in 100 ml of 80% aqueous methanol, and thereafter, 300 ml of normal-nucleic acid was added thereto, followed by repeated degreasing twice, and the lower layer was depressurized and concentrated.

이 추출물(0.7 g)을 메탄올 10ml로 녹인 후 그 중 1ml를 취하여 증류수로 10배 희석한 후 미리 메탄올과 증류수로 활성화시켜 놓은 Sep-pak C18 카트리지(워터스, 미국)에 흡착시킨 후 증류수로 세척한 다음 40% 및 80% 수용성 아세트니트릴 5ml로 각각 용리한 후 분취-고속액체크로마토그래피를 이용하여 40% 수용성 메탄올추출물로부터 트란스-레즈베라트롤-4'-O-베타-D-글루코사이드(분리시간, 16.8분) 11.8mg, 서프루티코솔 A(분리시간, 24.3분) 72mg과 서프루티코솔 B(분리시간, 25.5분) 68mg을 각각 분리하였다. 또한 상기와 동일한 방법으로 80% 수용성 메탄올 추출물로부터 트란스-레스베라트롤(분리시간, 23.4분) 7.6mg과 트란스-비니페린(trans-viniferin, 분자량 484, 분리시간, 28.2분] 45mg과 시스-비니페린(분자량 484, 분리시간 32.2분) 39mg을 각각 분리하였다.This extract (0.7 g) was dissolved in 10 ml of methanol, 1 ml of which was diluted 10-fold with distilled water, and then adsorbed onto a Sep-pak C18 cartridge (Waters, USA) previously activated with methanol and distilled water and washed with distilled water. And then eluted with 5 ml of 40% and 80% aqueous acetonitrile, respectively, and then trans-resveratrol-4'- O -beta-D-glucoside from the 40% aqueous methanol extract using preparative-high-performance liquid chromatography (separation time, 16.8 minutes) 11.8 mg, 72 mg of surfrutisol A (separation time, 24.3 minutes) and 68 mg of serruticosol B (separation time, 25.5 minutes) were separated. In the same manner as above, 7.6 mg of trans-resveratrol (separation time, 23.4 minutes) and trans-viniferin ( trans- viniferin, molecular weight 484, separation time, 28.2 minutes) 45 mg and cis-viniferin (80% aqueous methanol extract) Molecular weight 484, separation time 32.2 minutes) 39 mg were isolated.

이때 사용된 분취용 고속액체크로마토그래피의 분리조건은 다음과 같다:The separation conditions of the preparative high performance liquid chromatography used are as follows:

워터스델타프렙 4000 고속액체크로마토그래피;Waters Delta Prep 4000 Fast Liquid Chromatography;

칼럼, RCM Novapak C18(2.5센티미터 × 10센티미터);Column, RCM Novapak C 18 (2.5 cm × 10 cm);

자외선 검출기(UV280 & 308nm);Ultraviolet detectors (UV 280 & 308 nm);

속도, 5.0ml/분;Rate, 5.0 ml / min;

용매(용매 A, 0.1% 트리프로로초산 함유 10% 수용성 아세트니트릴 용액; 용매 B, 50% 수용성 아세트니트릴 용액, 용매 A --> 용매 B로 50분간 농도구배용출).Solvent (solvent A, 10% water soluble acetonitrile solution containing 0.1% triproroacetic acid; solvent B, 50% water soluble acetonitrile solution, solvent A-> concentration gradient elution with solvent B for 50 minutes).

그리고 이 때 분리된 6가지 화합물의 화학구조는 상기 발명의 상세한 설명에서 언급한 것이고, 분석용 고속액체크로마토그램 그래프는 도 1에, 그리고 이들 화합물의 상세한 자외선, 적외선, 핵자기공명 및 질량분광기 스펙트럼 데이터는 표 1 & 표 2에 각각 나타낸 바와 같았다.The chemical structures of the six compounds separated at this time are mentioned in the detailed description of the present invention, and the analytical high-speed liquid chromatogram graph is shown in FIG. 1 and detailed ultraviolet, infrared, nuclear magnetic resonance, and mass spectrometry spectra of these compounds. The data were as shown in Table 1 & Table 2, respectively.

이때 분석용 HPLC 조건은 다음과 같다.At this time, the analytical HPLC conditions are as follows.

칼럼, μBondapak C18(3.9cm ×30cm);Column, μBondapak C18 (3.9 cm × 30 cm);

용매 A(0.1% 트리프로로초산 함유 10% 수용성 아세트니트릴 용액) & 용매 B(50% 수용성 아세트니트릴 용액), 용매 A --> 용매 B로 50분간 농도구배용출;Gradient gradient elution with solvent A (10% water soluble acetonitrile solution containing 0.1% triproroacetic acid) & solvent B (50% water soluble acetonitrile solution), solvent A-> solvent B for 50 minutes;

자외선 검출기(UV280(A) & 308nm(B));Ultraviolet detectors (UV 280 (A) & 308nm (B) );

속도, 1.0ml/분.Speed, 1.0 ml / min.

1, 트란스-레즈베라트롤-4'-O-베타-D-글루코사이드; 1 , trans-resveratrol-4′- O -beta-D-glucoside;

2, 트란스-레즈베라트롤; 2 , trans-resveratrol;

3, 서프루티코솔 A; 3 , surfrutisol A;

4, 서프루티코솔 B; 4 , surfuticosol B;

5, 트란스-비니페린; 5 , trans-viniferin;

6, 시스-비니페린. 6 , cis-viniferin .

<표 1> :작약씨로부터 분리된 레즈베라트롤 및 그 유도체의 자외선 및 적외선분광기 스펙트럼 데이터 Table 1: Spectral data of ultraviolet and infrared spectroscopy of resveratrol and its derivatives isolated from peony seeds

Instrumental analysisInstrumental analysis UVλmaxnm(logε)UV λmax nm (log ε ) IRνmax(cm-1)IR νmax (cm -1 ) trans-ResveratrolResveratrol-4'-O-β-D-glucopyranosidetrans-ε-Viniferincis-ε-ViniferinSuffruticosol ASuffruticosol B trans -ResveratrolResveratrol-4'- O -β-D-glucopyranoside trans - ε -Viniferin cis - ε -ViniferinSuffruticosol ASuffruticosol B 219(4.28), 308(4.02),320(3.34)219(4.28), 308(4.02),320(3.34)218(4.52), 312(4.29),324(4.32)218(s), 287(s), 308(s),325(4.38)225(4.08), 282(3.42)225(4.10), 282(3.45)219 (4.28), 308 (4.02), 320 (3.34) 219 (4.28), 308 (4.02), 320 (3.34) 218 (4.52), 312 (4.29), 324 (4.32) 218 (s), 287 (s ), 308 (s), 325 (4.38) 225 (4.08), 282 (3.42) 225 (4.10), 282 (3.45) 3200-3300, 1587, 1512,1149, 9643200-3300, 1589, 1507,1150, 9663230, 1594, 1510, 1440,1002, 9643230, 1594, 1510, 1440,1002, 9643000-3600, 1600, 1500,1440, 1330, 1230,1160, 1000, 8303000-3600, 1600, 1500,1440, 1330, 1230,1160, 1000, 8303200-3300, 1587, 1512,1149, 9643200-3300, 1589, 1507,1150, 9663230, 1594, 1510, 1440,1002, 9643230, 1594, 1510, 1440,1002, 9643000-3600, 1600, 1500,1440, 1330, 1230,1160, 1000, 8303000-3600, 1600, 1500,1440, 1330, 1230,1160, 1000, 830

*시료의 자외선 흡수스펙트럼은 메탄올 용액으로 녹인 후 측정함. * UV absorption spectrum of the sample is measured after melting with methanol solution.

*시료의 적외선 흡수스펙트럼은 KBr plate를 이용하여 측정함. * The infrared absorption spectrum of the sample is measured by using KBr plate.

<표 2> :작약씨로부터 분리된 레즈베라트롤 및 그 유도체의 핵자기공명(in CD 3 OD) 및 질량분광기 스펙트럼 데이터 Table 2: Nuclear Magnetic Resonance (in CD 3 OD) and Mass Spectrometer Spectrum Data of Resveratrol and Its Derivatives Isolated from Peony Seeds

Instrumental analysisInstrumental analysis trans-Resveratrol trans -Resveratrol Resveratrol-4'-O-β-D-glucopyranosideResveratrol-4'- O -β-D-glucopyranoside 1H-NMRH-2H-4H-6H-2'H-3'H-5'H-6'HαHβSugar moietyH-1H-2H-3H-4H-5H-6aH-6b 1 H-NMRH-2H-4H-6H-2'H-3'H-5'H-6'HαHβSugar moietyH-1H-2H-3H-4H-5H-6aH-6b 6.43 (d,J=2.5 Hz)6.14 (t,J=2.5 Hz)6.43 (d,J=2.5 Hz)7.33 (d,J=8.5 Hz)6.75 (d,J=8.5 Hz)6.75 (d,J=8.5 Hz)7.33 (d,J=8.5 Hz)6.79 (d,J=16.5 Hz)6.94 (d,J=16.5 Hz) 6.43 (d, J = 2.5 Hz ) 6.14 (t, J = 2.5 Hz) 6.43 (d, J = 2.5 Hz) 7.33 (d, J = 8.5 Hz) 6.75 (d, J = 8.5 Hz) 6.75 (d, J = 8.5 Hz) 7.33 ( d , J = 8.5 Hz) 6.69 ( d , J = 16.5 Hz) 6.14 ( d , J = 16.5 Hz) 6.46 (d,J=2.0 Hz)6.18 (t,J=2.0 Hz)6.46 (d,J=2.0 Hz)7.43 (d,J=8.5 Hz)7.07 (d,J=8.5 Hz)7.07 (d,J=8.5 Hz)7.43 (d,J=8.5 Hz)6.87 (d,J=16.0 Hz)6.98 (d,J=16.0 Hz)4.92 (d,J 1,2=7.5 Hz)3.41 (t,J 2,3=7.8 Hz)3.44 (m,J 3,4=8.4 Hz)3.30 (t,J 4,5=8.4 Hz)3.47 (m,J 5,6a=2.6 Hz,J 5,6b=5.7 Hz)3.90 (dd,J 6a,6b=12.5 Hz)3.71 (dd,J 6a,6b=12.5 Hz) 6.46 (d, J = 2.0 Hz ) 6.18 (t, J = 2.0 Hz) 6.46 (d, J = 2.0 Hz) 7.43 (d, J = 8.5 Hz) 7.07 (d, J = 8.5 Hz) 7.07 (d, J = 8.5 Hz) 7.43 ( d , J = 8.5 Hz) 6.07 ( d , J = 16.0 Hz) 6.98 ( d , J = 16.0 Hz) 4.92 ( d , J 1,2 = 7.5 Hz) 3.41 ( t , J 2, 3 = 7.8 Hz) 3.44 ( m , J 3,4 = 8.4 Hz) 3.30 ( t , J 4,5 = 8.4 Hz) 3.47 ( m , J 5,6a = 2.6 Hz, J 5,6b = 5.7 Hz) ( dd , J 6a, 6b = 12.5 Hz) 3.71 ( dd , J 6a, 6b = 12.5 Hz) 13C-NMRC-1C-2C-3C-4C-5C-6CαCβC-1'C-2'C-3'C-4'C-5'C-6'Sugar moietyG-1G-2G-3G-4G-5G-6 13 C-NMRC-1C-2C-3C-4C-5C-6CαCβC-1'C-2'C-3'C-4'C-5'C-6'Sugar moietyG-1G-2G-3G-4G- 5G-6 141.31105.76159.37102.64159.37108.20127.02130.42131.40129.38115.84158.37116.48128.79141.31105.76159.37102.64159.37108.20127.02130.42131.40129.38115.84158.37116.48128.79 141.01105.95159.66102.95159.66105.95128.52128.88133.18128.58117.90158.65117.90128.58102.2174.9078.1371.3677.9762.50141.01105.95159.66102.95159.66105.95128.52128.88133.18128.58117.90158.65117.90128.58102.2174.9078.1371.3677.9762.50 FABMS (m/z)FABMS ( m / z ) 228[M+]228 [M + ] 390[M+]390 [M + ]

Coupling constants (Jin Hz) in parentheses.d, doublet;dd, double doublet;t, triplet;m, multiplet.Coupling constants ( J in Hz) in parentheses. d , doublet; dd , double doublet; t , triplet; m , multiplet.

trans-ε-Viniferin trans - ε -Viniferin cis-ε-Viniferin cis - ε -Viniferin Suffruticosol ASuffruticosol a Suffruticosol BSuffruticosol B 1H-NMRH-2 & H-6 7.14 (d,J=8.5)H-3 & H-5 6.76 (d,J=8.5)H-7 5.36 (d,J=5.5)H-8 4.34 (d,J=5.5)H-10 6.24 (d,J=2.0)H-12 6.16 (t,J=2.0)H-14 6.24 (d,J=2.0)H-2' 7.03 (d,J=8.5)H-3' 6.64 (d,J=8.5)H-5' 6.64 (d,J=8.5)H-6' 7.03 (d,J=8.5)H-7' 6.81 (d,J=16.5)H-8' 6.56 (d,J=16.5)H-12' 6.24 (d,J=2.0)H-14' 6.17 (d,J=2.0H-2" & H-6"H-3" & H-5"H-7"H-8"H-10"H-12"H-14" 1 H-NMRH-2 & H-6 7.14 ( d , J = 8.5) H-3 & H-5 6.76 ( d , J = 8.5) H-7 5.36 ( d , J = 5.5) H-8 4.34 ( d , J = 5.5) H-10 6.24 ( d , J = 2.0) H-12 6.16 ( t , J = 2.0) H-14 6.24 ( d , J = 2.0) H-2 '7.03 ( d , J = 8.5) H-3 '6.64 ( d , J = 8.5) H-5' 6.64 ( d , J = 8.5) H-6 '7.03 ( d , J = 8.5) H-7' 6.81 ( d , J = 16.5) H- 8 '6.56 ( d , J = 16.5) H-12' 6.24 ( d , J = 2.0) H-14 '6.17 ( d , J = 2.0H-2 "&H-6" H-3 "& H-5 "H-7" H-8 "H-10" H-12 "H-14" 6.93 (d,J=8.5)6.70 (d,J=8.5)5.17 (d,J=6.0)3.77 (d,J=2.0)5.92 (d,J=2.0)6.08 (t,J=2.0)5.92 (d,J=2.0)6.92 (d,J=8.5)6.57 (d,J=8.5)6.57 (d,J=8.5)6.92 (d,J=8.5)6.19 (d,J=12.0)6.02 (d,J=12.0)6.21 (d,J=2.0)6.23 (d,J=2.0)6.93 ( d , J = 8.5) 6.70 ( d , J = 8.5) 5.17 ( d , J = 6.0) 3.77 ( d , J = 2.0) 5.92 ( d , J = 2.0) 6.08 ( t , J = 2.0) 5.92 ( d , J = 2.0) 6.92 ( d , J = 8.5) 6.57 ( d , J = 8.5) 6.57 ( d , J = 8.5) 6.92 ( d , J = 8.5) 6.19 ( d , J = 12.0) 6.02 ( d , J = 12.0) 6.21 ( d , J = 2.0) 6.23 ( d , J = 2.0) 7.12 (d,J=8.5)6.73 (d,J=8.5)5.71 (d,J=11.5)4.34 (d,J=11.5)6.29 (d,J=2.0)5.95 (d,J=2.4)6.47 (d,J=8.5)6.14 (d,J=8.5)6.14 (d,J=8.5)6.47 (d,J=8.5)5.40 (d,J=3.2)3.90 (m)6.24 (s)6.96 (d,J=8.8)6.39 (d,J=8.8)3.69 (d,J=7.6)4.75 (s)6.01 (d,J=2.2)6.10 (t,J=2.2)6.00 (d,J=2.2)7.12 ( d , J = 8.5) 6.73 ( d , J = 8.5) 5.71 ( d , J = 11.5) 4.34 ( d , J = 11.5) 6.29 ( d , J = 2.0) 5.95 ( d , J = 2.4) 6.47 ( d , J = 8.5) 6.14 ( d , J = 8.5) 6.14 ( d , J = 8.5) 6.47 ( d , J = 8.5) 5.40 ( d , J = 3.2) 3.90 ( m ) 6.24 ( s ) 6.96 ( d , J = 8.8) 6.39 ( d , J = 8.8) 3.69 ( d , J = 7.6) 4.75 ( s ) 6.01 ( d , J = 2.2) 6.10 ( t , J = 2.2) 6.00 ( d , J = 2.2) 7.58 (d,J=8.5)6.91 (d,J=8.5)5.86 (d,J=11.5)5.09 (d,J=11.5)6.19 (d,J=2.0)5.96 (d,J=2.4)6.92 (brd)6.52 (d,J=8.0)6.52 (d,J=8.0)6.92 (brd)4.23 (d,J=11.7)4.12 (m)6.27 (s)6.28 (d,J=8.8)6.30 (d,J=8.8)3.82 (d,J=6.0)4.11 (s)6.24 (s)6.16 (t,J=2.2)6.23 (s)7.58 ( d , J = 8.5) 6.91 ( d , J = 8.5) 5.86 ( d , J = 11.5) 5.09 ( d , J = 11.5) 6.19 ( d , J = 2.0) 5.96 ( d , J = 2.4) 6.92 ( brd ) 6.52 ( d , J = 8.0) 6.52 ( d , J = 8.0) 6.92 ( brd ) 4.23 ( d , J = 11.7) 4.12 ( m ) 6.27 ( s ) 6.28 (d, J = 8.8) 6.30 (d, J = 8.8) 3.82 (d, J = 6.0) 4.11 ( s ) 6.24 ( s ) 6.16 ( t , J = 2.2) 6.23 ( s )

FABMS (m/z)454[M+]FABMS (m / z) 454 [M +] 454[M+]454 [M +] 680[M+]680 [M +] 680[M+]680 [M +]

13C-NMRC-1 133.79C-2 & C-6 128.21C-3 & C-5 116.31C-4 158.55C-7 94.71C-8 57.72C-9 148.25C-10 109.34C-11 159.77C-12 101.76C-13 159.83C-14 109.12C-1' 130.71C-2' 128.78C-3' 116.39C-4' 158.55C-5' 116.39C-6' 128.78C-7' 130.22C-8' 123.72C-9' 136.93C-10' 119.31C-11' 163.01C-12' 96.86C-13' 160.06C-14' 104.35C-1"C-2" & C-6"C-3" & C-5"C-4"C-7"C-8"C-9"C-10"C-11"C-12"C-13"C-14" 13 C-NMRC-1 133.79C-2 & C-6 128.21C-3 & C-5 116.31C-4 158.55C-7 94.71C-8 57.72C-9 148.25C-10 109.34C-11 159.77C-12 101.76C-13 159.83C-14 109.12C-1 '130.71C-2' 128.78C-3 '116.39C-4' 158.55C-5 '116.39C-6' 128.78C-7 '130.22C-8' 123.72C -9 '136.93C-10' 119.31C-11 '163.01C-12' 96.86C-13 '160.06C-14' 104.35C-1 "C-2"& C-6 "C-3"& C-5 "C-4" C-7 "C-8" C-9 "C-10" C-11 "C-12" C-13 "C-14" 133.84128.50116.20157.8294.9157.73147.33107.24159.53101.82159.53107.24130.07131.13115.88158.39115.88131.13131.63126.63137.81120.37162.7896.58159.53108.91133.84128.50116.20157.8294.9157.73147.33107.24159.53101.82159.53107.24130.07131.13115.88158.39115.88131.13131.63126.63137.81120.37162.7896.58159.53108.91 129.83129.11115.12157.3390.2347.93140.32125.61153.37100.78155.03104.69132.52129.41112.87152.87112.87129.4138.3747.61143.14115.85158.5995.04153.67121.74134.18129.22114.14154.7459.4653.17147.08105.61157.69100.26157.65105.69129.83129.11115.12157.3390.2347.93140.32125.61153.37100.78155.03104.69132.52129.41112.87152.87112.87129.4138.3747.61143.14115.85158.5995.04153.67121.74134.18129.22114.14154.7459.4653.17147.08105.61157.69100.26157.65105.69 129.46129.08115.12157.6789.7148.31141.02121.51155.77103.57156.87102.31132.42131.71113.37154.82113.37131.7145.0746.38146.04117.05159.8394.87154.27122.15134.05128.06113.78154.6461.6755.51146.03106.01158.09100.06158.04106.02129.46129.08115.12157.6789.7148.31141.02121.51155.77103.57156.87102.31132.42131.71113.37154.82113.37131.7145.0746.38146.04117.05159.8394.87154.27122.15134.05128.06113.78154.6461.6755.51146.03106.01156.09106.06.06.08.08.0. FABMS (m/z)454[M+]FABMS (m / z) 454 [M +] 454[M+]454 [M +] 680[M+]680 [M +] 680[M+]680 [M +]

s, singlet;d, doublet;dd, double doublet;m, multiplet;brd, broad. s , singlet; d , doublet; dd , double doublet; m , multiplet; brd , broad.

＜실험예 1＞: 작약씨로부터 분리된 레즈베라트롤 및 그 유도체의 항암 및 항돌연변이 작용 Experimental Example 1 Anticancer and Antimutagenic Activities of Resveratrol and Its Derivatives Isolated from Peony Seeds

제1단계: 세포독성 실험 Step 1 : Cytotoxicity Test

본 실험예에서 상기 실시예 1, 2, 3에서 얻은 레스베라트롤 및 그 유도체의 세포독성효과를 측정하기위해서 5가지 인체 암세포주[간암세포인 HepG2(human hepatocellular carcinoma), 유방암세포인 MCF7(human breast adenocarcinoma pleual effusion), 자궁경부암세포인 HeLa(human cervicse adenocarcinoma), 대장암 HT-29(human colon adenocarcinoma) 및 신경교종세포인 C6(mouse glioma)]를 1999년 3월 대전 소재 한국과학기술연구원 생명공학연구소로부터 분양받아 37℃, 5% CO2배양기에서 배양하면서 측정하였다.In order to measure the cytotoxic effect of the resveratrol and its derivatives obtained in Examples 1, 2 and 3 in the present experimental example, five human cancer cell lines (human hepatocellular carcinoma (HepG2) and breast cancer cells MCF7 (human breast adenocarcinoma) pleual effusion), cervical cancer cell HeLa (human cervicse adenocarcinoma), colorectal cancer HT-29 (human colon adenocarcinoma) and glioma cell C6 (mouse glioma)], March 1999 Daejeon Biotechnology Research Institute Was measured while incubating in a 37 ℃, 5% CO 2 incubator.

위의 5종의 암세포(HepG2, MCF7, HeLa, HT-26 및 C6)는 일주일에 2∼3회 새로운 배지로 교환하고 PBS 완충액(pH 7.0)로 세척한 후, 0.05% 티로신, 0.002% 이디티에이를 사용하여 부착된 세포를 분리하여 원심분리한 후, 집적된 암세포에 배지를 넣고 암세포가 골고루 분산되도록 피펫으로 잘 혼합하여 75센티미터 세포배양 플라스크에 10밀리리터씩 일정량 분할하여 주입하고, 4∼5일마다 계대 배양하면서 실험에 사용하였다.The above five cancer cells (HepG2, MCF7, HeLa, HT-26 and C6) were exchanged with fresh medium 2-3 times a week and washed with PBS buffer (pH 7.0), followed by 0.05% tyrosine, 0.002% editier Using this, the attached cells were separated and centrifuged, and then, the culture medium was added to the integrated cancer cells, mixed well by pipette to distribute the cancer cells evenly, and then injected into a 75 centimeter cell culture flask by dividing a predetermined amount by 10 ml, and 4 to 5 days. Each passage was used for the experiment while subcultured.

다음, 각 화합물의 세포독성(cytotoxicity)은 전보 (Han et al.,J. Kor. Soc. Food Sci. Nutr., 29: 153-160, 2000)와 같이 엠티티[MTT; 3-(4,5-dimethyl- thiazol-2-yl)-2,5-diphenyltetrazolium bromide] 에세이(Michael et al.,CancerRes., 48: 589-595, 1988; Carmichael et al.,Cancer Res., 47: 936-942, 1987)를 사용하여 측정하였다. 각 세포주(1×105세포/ml)를 24-웰프레이트(well plate)에 1ml씩 분주하고 24시간 동안 배양(37℃, 5% CO2) 한 후 무처리구인 control 군에는 디메틸설폭사이드(dimethylsulfoxide, DMSO)로 독성실험을 실시하였고, 각각의 시료는 DMSO에 녹여 100, 200, 300, 400 및 500㎍/웰의 농도로 첨가하여 48시간 동안 다시 배양하였다. 이 배양액에 엠티티 용액을 각각 100㎛씩 첨가하여 4시간 동안 배양시킨 후 웰 바닥에 형성된 포마잔(formazan)이 흩어지지 않게 조심스럽게 다루어 아스피레이트로 상등액을 제거하였다. 형성된 포마잔에 DMSO와 EtOH(1:1, v/v) 혼합용액 1ml 첨가하여 충분히 녹인 후 자외선-가시광선 분광광도계(UV-vis spectrophotometer, Pharmacia Biotech 80-1205-20, Sweden)를 이용하여 570nm에서 흡광도를 측정하여 재현성 여부를 검토한 후 첨가 시료의 암세포주에 대한 세포독성 여부를 비교 분석한 결과는 하기 표 3에 나타내었다. 위의 항암실험은 3구 3회 반복으로 실험하여 평균값으로 나타내었으며, 이때 표준오차는 생략하였다.Next, the cytotoxicity of each compound was reported by Han et al.,J. Kor. Soc. Food Sci. Nutr, 29: 153-160, 2000). 3- (4,5-dimethyl- thiazol-2-yl) -2,5-diphenyltetrazolium bromide] assay (Michael et al.,Cancer Res., 48: 589-595, 1988; Carmichael et al.,Cancer res, 47: 936-942, 1987). Each cell line (1 × 105Cells / ml) were dispensed in 1-well 24-well plates and incubated for 24 hours (37 ° C, 5% CO).2After treatment, the control group, which was not treated, was subjected to toxicity test with dimethylsulfoxide (DMSO), and each sample was dissolved in DMSO and added at a concentration of 100, 200, 300, 400, and 500 µg / well for 48 hours. Incubated again. The culture solution was incubated for 4 hours by adding 100 µm each of the empty solution to the culture solution, and the supernatant was removed by aspirate by carefully treating the formazan formed on the bottom of the well so as not to scatter. 1 ml of mixed solution of DMSO and EtOH (1: 1, v / v) was added to the formed formazan, which was then sufficiently dissolved. After examining the reproducibility by measuring the absorbance at and the comparative analysis of the cytotoxicity to the cancer cell line of the added sample is shown in Table 3 below. The above anticancer test was repeated three times and three times to represent the average value, and the standard error was omitted.

<표 3> : 작약씨로부터 분리된 레즈베라트롤 및 그 유도체의 세포독성 효과TABLE 3 Cytotoxic Effects of Resveratrol and Its Derivatives Isolated from Peony Seeds

세포주Cell line 농도(μg/mL)Concentration (μg / mL) 억제율(%)* % Suppression * 화합물1 Compound 1 화합물2 Compound 2 화합물3 Compound 3 화합물4 Compound 4 화합물5 Compound 5 화합물6 Compound 6 HeLa(자궁경부암)HeLa (cervical cancer) 2020 27.427.4 8.58.5 48.548.5 21.921.9 9.59.5 9.89.8 4040 27.827.8 28.428.4 79.179.1 44.744.7 8.78.7 8.38.3 6060 29.229.2 75.675.6 86.286.2 78.378.3 8.48.4 5.45.4 8080 29.729.7 94.894.8 94.794.7 84.884.8 6.26.2 3.23.2 100100 28.928.9 98.698.6 95.295.2 91.291.2 15.215.2 11.211.2 MCF-7(유방암)MCF-7 (breast cancer) 2020 4.54.5 18.218.2 2.22.2 81.281.2 3.43.4 5.15.1 4040 8.68.6 21.721.7 35.635.6 87.987.9 4.84.8 4.24.2 6060 19.319.3 69.369.3 74.774.7 90.290.2 1.41.4 -4.5-4.5 8080 20.120.1 94.494.4 91.491.4 95.495.4 -3.2-3.2 -9.2-9.2 100100 19.719.7 96.896.8 92.392.3 97.897.8 -5.7-5.7 -11.5-11.5 C6(신경교종암)C6 (neuroglioma) 2020 63.363.3 95.495.4 54.354.3 74.374.3 45.345.3 58.358.3 4040 72.172.1 99.299.2 83.183.1 85.385.3 66.766.7 62.162.1 6060 78.378.3 99.899.8 92.192.1 86.286.2 71.471.4 68.368.3 8080 81.281.2 99.999.9 93.193.1 87.287.2 78.278.2 69.269.2 100100 82.682.6 99.999.9 98.298.2 95.495.4 80.280.2 70.270.2 HepG2(간암)HepG2 (liver cancer) 2020 9.29.2 18.118.1 14.214.2 78.378.3 18.418.4 18.418.4 4040 12.112.1 27.227.2 28.528.5 89.289.2 16.616.6 19.319.3 6060 13.313.3 78.478.4 61.761.7 94.594.5 22.222.2 20.220.2 8080 18.218.2 99.199.1 82.782.7 99.399.3 29.629.6 22.322.3 100100 21.621.6 99.899.8 92.592.5 99.899.8 56.456.4 52.652.6 HT-29(대장암)HT-29 (Colon Cancer) 2020 8.38.3 25.625.6 14.414.4 14.314.3 16.916.9 13.513.5 4040 16.716.7 67.267.2 18.218.2 29.729.7 22.722.7 15.715.7 6060 39.939.9 73.573.5 31.731.7 34.634.6 38.238.2 34.234.2 8080 52.352.3 83.783.7 63.763.7 69.369.3 51.351.3 42.342.3 100100 68.768.7 93.393.3 80.880.8 81.581.5 67.567.5 61.761.7

*CControl구에 대한 저해율. * Inhibition of the CControl sphere.

화합물1, 트란스-레즈베라트롤-4'-O-베타-D-글루코사이드;Compound 1 , trans-resveratrol-4'- 0 -beta-D-glucoside;

화합물2, 트란스-레즈베라트롤 ;Compound 2 , trans-resveratrol;

화합물3, 트란스-비니페린;Compound 3 , trans-viniferin;

화합물4, 시스-비니페린;Compound 4 , cis-viniferin;

화합물5, 서프루티코솔 A;Compound 5 , surfrutisol A;

화합물6, 서프루티코솔 B.Compound 6 , surfrutisol B.

상기 표 3에 보는 바와 같이 작약씨로부터 분리된 6가지 스틸벤화합물(stilbenes)을 플레이트당 20, 40, 60, 80, 100㎍의 농도로 첨가하여 항암효과를 측정한 결과 모든 암세포에 대해 상당한 세포독성 효과를 나타내었으며, 특히 신경교종 암세포에 대해 가장 강한 암세포사멸 효과를 나타내었다. 그리고 5가지 화합물 중 트란스-레즈베라트롤은 모든 암세포에 대해 가장 강한 항암작용을 나타내었으며, 아울러 트란스-비니페린과 시스-비니페린도 공히 모든 암세포에 대해 높은 항암작용을 나타내었다. 그러나 트란스-레즈베라트롤-4'-O-베타-D-글루코사이드 및 서프루티코솔 A & B는 모든 암세포에 대해 세포독성효과가 미약하였다.As shown in Table 3, six stilbenes isolated from peony seeds were added at 20, 40, 60, 80, and 100 ㎍ concentrations per plate, and the anticancer effect was measured. It showed a toxic effect, and particularly showed the strongest cancer cell death effect against glioma cancer cells. Of the five compounds, trans-resveratrol showed the strongest anticancer activity against all cancer cells, and trans-viniferin and cis-viniferin also showed high anticancer activity against all cancer cells. However, trans-resveratrol-4'- O -beta-D-glucoside and surfrucosol A & B had a weak cytotoxic effect on all cancer cells.

제2단계: 돌연변이원성 및 항돌연변이원성 실험 Phase 2 : Mutagenicity and Antimutagenicity

작약씨로부터 분리된 레즈베라트롤 및 그 유도체의 돌연변이 및 항돌연변원성 실험은 에임즈(Ames) 테스트를 개량한 프리인큐베이션(preincubation) 방법(Martin and Martin,Cytotechnology11: 49-52. 1997)에 따라 히스티딘 영양요구주로서 변이주(point mutant)인 살모넬라 티피머륨(Salmonella typhimurium) TA100(hisG 46, rfa, △uvrB)을 사용하여 His+복귀 돌연변이 정도를 조사하였다.Mutation and antimutagenicity experiments of resveratrol and its derivatives isolated from Peony seeds were performed according to histidine according to the preincubation method (Martin and Martin, Cytotechnology 11: 49-52, 1997) that improved the Ames test. The extent of His + return mutation was investigated using Salmonella typhimurium TA100 (hisG 46, rfa, ΔuvrB), a mutant point mutant.

항돌연변이원성 실험에 사용된 돌연변이원은 엠엔엔지[MNNG; N-methyl-N-nitro-N-nitrosoguanidine)를 프레이트당 5㎍으로 조정하여 사용하였다. 미리 건열 멸균시킨 유리캡 튜브에 시료 용액(농도 표기)을 0.05ml를 첨가하고 변이원을 0.05ml를 첨가한 다음 여기에 영양배지에서 16시간 배양시킨 살모넬라 티피머륨 TA100 배양액 0.1ml와 0.2몰 나트륨인산완충액(pH 7.4) 0.5ml를 첨가하였다. 이것을 37℃에서 30분간 배양한 다음 소량의 히스티딘/비오틴이 첨가된 아가(top agar, 45℃) 3ml를 혼합한 후 미리 조제해 놓은 최소 포도당 한천배지 프레이트상에 도말하고 평판 고화시킨 다음 37℃에서 48시간 배양하였다. 항돌연변이 활성은 상기의 고체배지에서 생육하는 His+복귀 돌연변이 콜로니 수를 측정한 후 아래식에 따라 His+복귀돌연변이 저해율을 산출하였다.Mutants used in antimutagenicity experiments include MNNG; N-methyl-N-nitro-N-nitrosoguanidine) was used at 5 μg per plate. To the dry heat sterilized glass cap tube, 0.05 ml of the sample solution (concentration notation) was added, 0.05 ml of the mutagen was added, and 0.1 ml of Salmonella typhimerium TA100 culture cultured in a nutrient medium for 16 hours and 0.2 mol sodium phosphate. 0.5 ml of buffer (pH 7.4) was added. This was incubated for 30 minutes at 37 ° C, then a small amount of histidine / biotin-added 3 ml of agar (45 ° C) was added, plated on a pre-prepared minimum glucose agar medium plate and solidified at 37 ° C. Incubated for 48 hours. Antimutagenic activity was measured by the number of His + return mutant colonies growing in the solid medium, and the inhibition rate of His + return mutation was calculated according to the following equation.

저해율(%) = [(a-b)/(a-c)] ×100% Inhibition = [(a-b) / (a-c)] × 100

a: 변이원에 의해 유도된 His+복귀 돌연변이 콜로니 수a: Number of His + return mutant colonies induced by mutagens

b: 변이원과 시료 처리시 유도된 His+복귀 돌연변이 콜로니 수b: Number of His + return mutant colonies induced in mutagen and sample processing

c: 변이원과 시료 무처리시 유도된 His+복귀 돌연변이 콜로니 수c: Number of His + return mutant colonies induced in mutagen and sample untreated

한편, 분리된 화합물의 돌연변이원성 유무를 조사하기위해 변이원을 첨가하지 않고 시료만을 첨가하여 상기의 항돌연변이원성 실험과 같은 방법으로 실시하였다. 돌연변이활성은 시료에 의한 His+복귀 돌연변이율로서 무처리시 유도된 His+복귀 돌연변이 콜로니 수(자연 복귀 돌연변이 콜로니 수)에 대한 시료 처리시 유도된 His+복귀 돌연변이 콜로니 수의 %로 나타내었다. 위의 돌연변이 및 항돌연변이 실험은 3구 3회 반복으로 실험하여 평균값으로 표 4에 나타내었으며, 이때 표준오차는 생략하였다.On the other hand, in order to examine the presence or absence of mutagenicity of the isolated compound was carried out in the same manner as the above anti-mutagenicity experiment by adding only the sample without adding a mutagen. Mutagenic activity was expressed as% of His + return mutant colonies derived at sample treatment versus His + return mutant colonies (natural return mutant colonies) induced at no treatment as His + return mutation rate by sample. The above mutation and antimutation experiments were performed in three replicates three times and the average values are shown in Table 4, and the standard error was omitted.

<표 4> : 작약씨로부터 분리된 레즈베라트롤 및 그 유도체의 돌연변이 효과Table 4: Mutagenic Effects of Resveratrol and Its Derivatives Isolated from Peony Seeds

화합물compound 농도(μg/plate)Concentration (μg / plate) His+revertant 수/plateHis + revertant number / plate 돌연변이율(%)% Mutation 화합물1 Compound 1 102050100102050100 4348464543484645 0.8961.0000.9580.9380.8961.0000.9580.938 화합물2 Compound 2 102050100102050100 4649454246494542 0.9581.0210.9380.8750.9581.0210.9380.875 화합물3 Compound 3 102050100102050100 4048454340484543 0.8331,0000.9380.8960.8331,0000.9380.896 화합물4 Compound 4 102050100102050100 4445434844454348 0.9170.9380.8961.0000.9170.9380.8961.000 화합물5 Compound 5 102050100102050100 4943454849434548 1.0210.8960.9381.0001.0210.8960.9381.000 화합물6 Compound 6 102050100102050100 5149424551494245 1.0631.0210.8750.9381.0631.0210.8750.938 Negative controlNegative control 4848

화합물1, 트란스-레즈베라트롤-4'-O-베타-D-글루코사이드;Compound 1 , trans-resveratrol-4'- 0 -beta-D-glucoside;

화합물2, 트란스-레즈베라트롤 ;Compound 2 , trans-resveratrol;

화합물3, 트란스-비니페린;Compound 3 , trans-viniferin;

화합물4, 시스-비니페린;Compound 4 , cis-viniferin;

화합물5, 서프루티코솔 A;Compound 5 , surfrutisol A;

화합물6, 서프루티코솔 B.Compound 6 , surfrutisol B.

표 4에서 보는 바와 같이 작약씨로부터 분리된 6가지 화합물을 처리하지 않은 음성대조군의 복귀 돌연변이 집락수가 48이었으며, 각 화합물을 첨가한 처리구의 집락수가 음성대조군에 비하여 농도의존적으로 돌연변이율이 증가하지 않았으므로 모든 화합물은 돌연변이원성 및 독성을 나타내지 않았다.As shown in Table 4, the number of colonies returned from the negative control group without treatment of the six compounds isolated from the peony seeds was 48, and the number of colonies of the treatment groups to which each compound was added did not increase in the concentration-dependent mutation rate compared to the negative control group. All compounds did not show mutagenicity and toxicity.

<표 5> : 작약씨로부터 분리된 레즈베라트롤 및 그 유도체의 항돌연변이 효과Table 5: Antimutagenic Effects of Resveratrol and Its Derivatives Isolated from Peony Seeds

화합물compound 농도(μg/plate)Concentration (μg / plate) His+Revertants수/plateHis + Revertants number / plate 저해율(%)% Inhibition 화합물1 Compound 1 102050100102050100 10525674122591052567412259 13.7555.4168.7381.8713.7555.4168.7381.87 화합물2 Compound 2 102050100102050100 10066802251621006680225162 17.7045.7184.7990.2117.7045.7184.7990.21 화합물3 Compound 3 102050100102050100 10636804533321063680453332 12.8045.7165.2175.6012.8045.7165.2175.60 화합물4 Compound 4 102050100102050100 10777394383031077739438303 11.6040.8966.5078.0911.6040.8966.5078.09 화합물5 Compound 5 102050100102050100 10617023482141061702348214 12.9743.8174.2285.7412.9743.8174.2285.74 화합물6 Compound 6 102050100102050100 10567153602251056715360225 13.4042.7073.2084.7913.4042.7073.2084.79 Negative controlPositive controlNegative control 481212481212

화합물1, 트란스-레즈베라트롤-4'-O-베타-D-글루코사이드;Compound 1 , trans-resveratrol-4'- 0 -beta-D-glucoside;

화합물2, 트란스-레즈베라트롤 ;Compound 2 , trans-resveratrol;

화합물3, 트란스-비니페린;Compound 3 , trans-viniferin;

화합물4, 시스-비니페린;Compound 4 , cis-viniferin;

화합물5, 서프루티코솔 A;Compound 5 , surfrutisol A;

화합물6, 서프루티코솔 B.Compound 6 , surfrutisol B.

표 5에서 보는 바와같이 작약씨로부터 분리된 5가지 화합물을 플레이트당 10, 20, 50, 100μg의 농도로 첨가하여 항돌연변이 효과를 측정한 결과 모두 농도의존적으로 돌연변이 억제율이 증가함을 알 수 있었다. 특히, 5가지 화합물 중 트란스-레즈베라트롤이 가장 강한 돌연변이 억제 효과를 나타내었으며, 다음으로 레즈베라트롤 배당체인 트란스-레즈베라트롤-4'-O-베타-D-글루코사이드가 높은 효과를 보였다. 그리고 트란스-비니페린, 시스-비니페린, 및 서프루티코솔 A & B 성분도 비록 트란스-레즈베라트롤 보다 항돌연변이 효과가 낮았으나 상당한 돌연변이 억제 효과를 나타내었다. 이와같이 작약씨로부터 분리된 레즈베라트롤 및 그 유도체는 항암 및 항돌연변이 효과가 강함을 알 수 있었다.As shown in Table 5, five compounds isolated from peony seeds were added at concentrations of 10, 20, 50, and 100 μg per plate, and the antimutagenic effects were measured. In particular, trans-resveratrol showed the strongest mutation inhibitory effect among the five compounds, followed by trans-resveratrol-4'- O -beta-D-glucoside, which is the resveratrol glycoside. In addition, trans-viniferin, cis-viniferin, and surfrucosol A & B components showed a significant mutation inhibitory effect, although the antimutagenic effect was lower than that of trans-resveratrol. Thus, the resveratrol and its derivatives isolated from peony seeds were found to have strong anticancer and antimutagenic effects.

본 발명의 작약씨로부터 분리·동정된 트란스-레즈베라트롤, 트란스-레즈베라트롤-4'-글루코사이드, 비니페린(시스- & 트란스-비니페린) 및 서프루티코솔 A & B 성분인 레즈베라트롤 및 그 유도체는 높은 항암 및 항돌연변이 효과를 지니고 있으므로 암 예방용 및 치료용 의약품 및 기능성 식품의 신소재를 제공할 수 있다. 또한 이들 작약씨의 레즈베라트롤 및 그 유도체는 통상의 부형제와 함께 수액제, 환제, 정제, 캡슐 형태로 제조되어 암 예방용 및 치료제의 의약품 또는 식품첨가제와 함께 분말 또는 과립 차 및 음료와 같은 형태로 제조되어 기능성 건강보조식품으로도 사용될 수 있다.Resveratrol, which is a trans-resveratrol, trans-resveratrol-4'-glucoside, viniperin (cis- & trans-viniferin) and supruticosol A & B components isolated and identified from the peony seed of the present invention And since the derivative has a high anti-cancer and anti-mutagenic effect, it can provide a new material of medicines and functional foods for preventing and treating cancer. In addition, resveratrol and its derivatives of these peony seeds are prepared in the form of sap, pills, tablets, capsules with conventional excipients, in the form of powder or granule tea and beverage together with pharmaceutical or food additives for the prevention and treatment of cancer. It can also be used as a functional dietary supplement.

Claims (11)

건조된 작약씨를 미세하게 분쇄하여 노르말 핵산으로 탈지하여 지방을 제거하는 단계와,Finely pulverizing the dried peony seed and degreasing with normal nucleic acid to remove fat, 지방을 제거한 작약씨 잔사에 메탄올을 가하여 상온에서 추출한 후 감압·농축하는 단계와,Extracting at room temperature by adding methanol to the peony seed residue from which fat was removed, and decompressing and concentrating; 전기의 농축물을 다이아이온 HP-20로 칼럼크로마토그래피를 실시하여 추출물을 얻는 단계와,Performing the column chromatography with diion HP-20 on the former concentrate to obtain an extract, 전기의 추출물을 세파덱스 LH-20 칼럼 및 분취-고속액체크로마토그래피를 이용하여 분리한 후 재결정하는 단계를 포함하는 것을 특징으로 하는 작약씨로부터 트란스-레즈베라트롤 및 그 유도체의 추출방법Separation of the former extract using a Sephadex LH-20 column and preparative-high-performance liquid chromatography, and then recrystallization method comprising the step of extracting trans- resveratrol and its derivatives from peony seed 건조된 작약씨를 미세하게 분쇄한 후 메탄올로 추출한 후 감압·농축하는 단계와,Finely pulverizing the dried peony seed and extracting with methanol, followed by decompression and concentration; 메탄올추출물을 다시 메탄올로 가용화시킨 후 노르말 핵산으로 탈지하여 지방을 제거하는 단계와,Solubilizing the methanol extract with methanol and then degreasing with normal nucleic acid to remove fat, 전기의 메탄올추출물에 에테르 및 에틸아세테이트를 순차적으로 넣고 분획한 후 얻어지는 에테르 및 에틸아세테이트층을 감압·농축하는 단계와,Depressurizing and concentrating the ether and ethyl acetate layers obtained by sequentially adding and distilling ether and ethyl acetate into methanol extracts; 전기의 에테르 및 에틸아세테이트층을 순차적으로 실리카겔, 세파덱스 엘에이치-20 칼럼 및 분취용 고속액체크로마토그래피를 이용하여 분리한 후 재결정하는단계를 포함하는 것을 특징으로 하는 작약씨로부터 트란스-레즈베라트롤 및 그 유도체의 추출방법Trans-Resveratrol from Peony seed, comprising the step of separating the first ether and ethyl acetate layer by using silica gel, Sephadex L-20 column and preparative high-performance liquid chromatography sequentially Extraction method of the derivative 제 1 항에 있어서, 추출용매를 메탄올 대신 에탄올을 사용하는 것을 특징으로 하는 작약씨로부터 트란스-레즈베라트롤 및 그 유도체의 추출방법The method of extracting trans-resveratrol and its derivatives from peony seed according to claim 1, wherein ethanol is used instead of methanol as the extraction solvent. 제 1항 또는 제 2항에 있어서, 메탄올 농도가 40% 내지 100% 수용액임을 특징으로 하는 작약씨 트란스-레즈베라트롤 및 그 유도체의 추출방법The method of extracting Peony seed Trans-Resveratrol and its derivatives according to claim 1 or 2, wherein the methanol concentration is 40% to 100% aqueous solution. 제 1 항 또는 제 2 항에 있어서, 작약씨로부터 추출한 트란스-베즈베라트롤 및 그 유도체는 하기 구조식 Ⅰ의 트란스-베즈베라트롤(trans-Resveratrol), 구조식 Ⅱ의 트란스-레즈베라트롤-4'-글루코사이드(Resveratrol-4'-O-β-D-glucopyranoside), 구조식 Ⅲ의 트란스-비니페린(trans-ε-Viniferin), 구조식 Ⅳ의 시스-비니페린(cis-ε-Viniferin), 구조식 Ⅴ의 서프루티코솔 A(Suffruticosol A) 및 구조식 Ⅵ의 서프루티코솔 B(Suffruticosol B)임을 특징으로 하는 작약씨로부터 트란스-레즈베라트롤 및 그 유도체의 추출방법The method according to claim 1 or 2, wherein the trans- besveratrol and its derivatives extracted from Peony seed are trans- Resveratrol of formula I, trans-resveratrol-4'- of formula II Glucoside (Resveratrol-4'- O- β-D-glucopyranoside), trans - ε- Viniferin of Structural Formula III, cis - ε- Viniferin of Structural Formula IV, the book of Structural Formula V Extraction method of Trans-Resveratrol and its derivatives from Peony seed characterized in that it is Suffruticosol A and Suffruticosol B of Structural VI (구조식 I) (구조식 II)(Formula I) (Formula II) (구조식 III) (구조식 Ⅳ)(Structure III) (Structure IV) (구조식 Ⅴ) (구조식 Ⅵ)(Structure Formula Ⅴ) (Structure Formula Ⅵ) 작약씨로부터 추출한 트란스-레즈베라트롤, 트란스-레즈베라트롤-4'-글루코사이드, 트란스-비니페린, 시스-비니페린, 서프루티코솔 A 또는 서프루티코솔 B 중에서 어느 하나의 단일 화합물을 유효성분으로 함유하는 신경교종암(C6) 치료제A single compound of any one of Trans-Resveratrol, Trans-Resveratrol-4'-glucoside, Trans-Viniferin, Cis-Viniferin, Supruticosol A, or Supruticosol B extracted from Peony seeds as an active ingredient Containing glioma cancer (C6) 작약씨로부터 추출한 트란스-비니페린 또는 시스-비니페린 성분 중 어느 하나의 단일 화합물을 유효성분으로 함유하는 간암, 유방암, 대장암 또는 자궁암 치료제A therapeutic agent for liver cancer, breast cancer, colon cancer, or uterine cancer containing a single compound of either trans-viniferin or cis-viniferin extracted from peony seed as an active ingredient 작약씨로부터 추출한 트란스-레즈베라트롤, 트란스-레즈베라트롤-4'-글루코사이드, 트란스-비니페린, 시스-비니페린, 서프루티코솔 A 또는 서프루티코솔 B를 유효성분으로 함유하는 항돌연변이제Antimutagenic containing Trans-Resveratrol, Trans-Resveratrol-4'-glucoside, Trans-Viniferin, Cis-Viniferin, Serfruticosol A or Serfruticosol B extracted from Peony seeds as an active ingredient 암 예방제 또는 치료제에 있어서, 작약씨로부터 추출한 트란스-레즈베라트롤, 트란스-레즈베라트롤-4'-글루코사이드, 트란스-비니페린, 시스-비니페린, 서프루티코솔 A 또는 서프루티코솔 B를 유효성분으로 함유하는 수액제, 환제, 정제 또는 캡슐제In the preventive or therapeutic agent for cancer, trans-resveratrol, trans-resveratrol-4'-glucoside, trans-viniferin, cis-viniferin, serproticosol A, or serruticosol B extracted from Peony seeds are active ingredients. Fluids, pills, tablets or capsules 암 예방용 식품에 있어서, 작약씨로부터 추출한 트란스-레즈베라트롤, 트란스-레즈베라트롤-4'-글루코사이드, 트란스-비니페린, 시스-비니페린, 서프루티코솔 A 또는 서프루티코솔 B를 유효성분으로 함유한 분말차, 과립차 또는 음료In the food for cancer prevention, trans-resveratrol, trans-resveratrol-4'-glucoside, trans-viniferin, cis-viniferin, serruticosol A, or serruticosol B extracted from peony seeds are active ingredients. Powdered tea, granulated tea or beverage 암 예방용 식품에 있어서, 작약씨로부터 추출한 트란스-레즈베라트롤, 트란스-레즈베라트롤-4'-글루코사이드, 트란스-비니페린, 시스-비니페린, 서프루티코솔 A 또는 서프루티코솔 B를 유효성분으로 함유한 정제 또는 캡슐제 형태의 건강보조식품In the food for cancer prevention, trans-resveratrol, trans-resveratrol-4'-glucoside, trans-viniferin, cis-viniferin, serruticosol A, or serruticosol B extracted from peony seeds are active ingredients. Supplements in the form of tablets or capsules
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100681439B1 (en) * 2005-10-10 2007-02-15 박창은 Anti-diabetic and anti-obesity preparation
KR100804993B1 (en) * 2007-01-29 2008-02-20 경상북도 농업기술원 Manufacturing method of toilet soap contained the extracts from seeds of paeonia lactiflora p
KR100893788B1 (en) * 2007-10-11 2009-04-20 서원대학교산학협력단 Method for purification of resveratrol from extract of grape twig
CN103235070A (en) * 2013-05-14 2013-08-07 张家港威胜生物医药有限公司 High performance liquid chromatography method for determining content of resveratrol in polygonum cuspidatum
KR101538047B1 (en) * 2013-09-12 2015-07-21 부산대학교 산학협력단 Composition containing polysacchride-removed Paeonia lactiflora extract for improving pregnancy
KR20150097256A (en) * 2014-02-18 2015-08-26 동국대학교 산학협력단 Resveratrol multimers with selective inhibitory of genome replication of HCV and use thereof
CN118045096A (en) * 2024-04-07 2024-05-17 华南理工大学 Red peony monoterpene compound and application thereof

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100681439B1 (en) * 2005-10-10 2007-02-15 박창은 Anti-diabetic and anti-obesity preparation
KR100804993B1 (en) * 2007-01-29 2008-02-20 경상북도 농업기술원 Manufacturing method of toilet soap contained the extracts from seeds of paeonia lactiflora p
KR100893788B1 (en) * 2007-10-11 2009-04-20 서원대학교산학협력단 Method for purification of resveratrol from extract of grape twig
CN103235070A (en) * 2013-05-14 2013-08-07 张家港威胜生物医药有限公司 High performance liquid chromatography method for determining content of resveratrol in polygonum cuspidatum
KR101538047B1 (en) * 2013-09-12 2015-07-21 부산대학교 산학협력단 Composition containing polysacchride-removed Paeonia lactiflora extract for improving pregnancy
KR20150097256A (en) * 2014-02-18 2015-08-26 동국대학교 산학협력단 Resveratrol multimers with selective inhibitory of genome replication of HCV and use thereof
CN118045096A (en) * 2024-04-07 2024-05-17 华南理工大学 Red peony monoterpene compound and application thereof
CN118045096B (en) * 2024-04-07 2024-06-21 华南理工大学 Red peony monoterpene compound and application thereof

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